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CyQUANT™ LDH Cytotoxicity Assay (Invitrogen™)

The CyQUANT LDH Cytotoxicity Assay is a colorimetric assay that provides a simple and reliable method for determining cellular cytotoxicity. Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many different cell types that is released into the cell culture medium upon damage to the plasma membrane. The CyQUANT LDH Cytotoxicity Assay provides the reagents to accurately and quantitatively measure this extracellular LDH.

Note: The CyQUANT LDH Cytotoxicity Assay (Cat. Nos. C20300 and C20301) are direct replacements for the Pierce LDH Cytotoxicity Assay Kit (Cat. Nos. 88953 and 88954).

CyQUANT LDH Cytotoxicity Assay features include:
Convenient—add-mix-read assay format for adherent and suspension cells, including 3D cell models
Accurate—provides a quantitative measurement of LDH release
Flexible—ideal for high-throughput screening, monitor cytotoxicity from the same sample over time
Robust—uses stable LDH enzyme activity as a cytotoxic marker

LDH is a cytosolic enzyme present in many different cell types and is a well-established and reliable indicator of cellular toxicity. Damage of the plasma membrane results in a release of LDH into the surrounding cell culture medium. This extracellular LDH can be quantified by a coupled enzymatic reaction in which LDH catalyzes the conversion of lactate to pyruvate via NAD+ reduction to NADH. Diaphorase then uses NADH to reduce a tetrazolium salt (INT) to a red formazan product that can be measured at 490 nm. The level of formazan formation is directly proportional to the amount of LDH released into the medium.

The CyQUANT LDH Cytotoxicity Assay provides the reagents needed for the simple, reliable colorimetric quantification of cellular cytotoxicity. The kit can be used with different cell types, including 3D cell models, to measure cytotoxicity mediated by chemical compounds as well as cell-mediated cytotoxicity. Since LDH in the medium is the indicator of cellular cytotoxicity, the assay can be used to monitor cytotoxicity from the same sample over time. To perform the assay, an aliquot of the cell culture medium is transferred to a new plate and the reaction mixture is added. After a 30-minute incubation, the reaction is stopped by adding Stop Solution and absorbance is measured using a microplate reader.

APO BrdU (TUNEL) Kit (Invitrogen™)

The ApoTarget™ APO-BRDU Kit is a two-color staining method for labeling DNA breaks and total cellular DNA to detect apoptotic cells by flow cytometry.

View a selection guide for all apoptosis assays for flow cytometry.

pHrodo™ Red E. coli BioParticles™ Conjugate for Phagocytosis (Invitrogen™)

New pH-sensitive pHrodo® dye conjugates give faster and more accurate results than any other phagocytosis assay.  pHrodo® dye conjugates are non-fluorescent outside the cell, but fluoresce brightly red in phagosomes.  Get faster staining and more accurate results—without the need for wash steps or quencher dye.

• Specific detection of phagocytosis and endocytosis
• Reduced signal variability and improved timing in sensitive experiments.
• Multiplex with green dyes such as GFP, Fluo-4, or calcein

The fluorescence of the novel pHrodo® dye dramatically increases as pH decreases from neutral to acidic, making it an ideal tool to study phagocytosis and its regulation by drugs and/or environmental factors. The lack of fluorescence outside the cell eliminates the need for wash steps and quencher dyes. Use the ready-made pHrodo® E.coli BioParticles® conjugates in imaging or flow applications, or pHrodo® SE, the activated succinimidyl ester, for labeling microorganisms or proteins of your choice.

CaspGLOW™ Fluorescein Active Caspase-8 Staining Kit (Invitrogen™)

The CaspGLOW™ Fluorescein Active Caspase-8 Staining Kit contains all the reagents necessary to detect active caspase-8 in cells with high sensitivity. Fluorescein (FITC)-conjugated IETD-FMK, a specific inhibitor of caspase-8, is utilized in this assay for detection. This reagent is cell permeable, non-toxic and irreversibly binds to the active enzyme. Detection of the labeled cells can be determined by flow cytometry, fluorescent microscopy or a fluorescent plate reader.

The caspases constitute a family of aspartate-specific cysteine proteases that mediate a sequence of cleavage events. Recruitment of the inactive proenzyme to oligomerized receptors leads to caspase activation and autoproteolytic cleavage. These active enzymes can then cleave other caspases, thereby generating a caspase signaling cascade that leads to a form of programmed cell death termed apoptosis.

Caspase-8, which is also known as FLICE, MACHalpha1, and Mch5, cleaves its substrates at the C-terminal aspartic acid residue of the motif Asp-X-X-Asp. Eight isoforms of caspase-8 exist, of which caspase-8/a and 8/b are the predominant forms. Upon stimulation of death receptors such as CD95/APO-1/Fas, TRAIL-R1, TRAIL-R2, TNFR1, and TRAMP, caspase-8 is recruited to the death-inducing signaling complex (DISC). Subsequent dimerization leads to caspase-8 activation via autocatalytic cleavage, which leads to the formation of a 12-kDa prodomain and a 43-kDa intermediate fragment that is further cleaved to produce 26-kDa and 18-kDa active enzymes. As an initiator caspase, this enzyme initiates a caspase signaling cascade that results in apoptosis. Substrates of caspase-8 include caspases-3 and -7, as well as the pro-apoptotic Bcl-2 family member Bid. In addition to its role in cell death, caspase-8 has been linked to cell adhesion and motility.

Reported Application
Flow Cytometric Analysis, Immunocytochemistry

Apoptotic DNA Ladder Kit (Invitrogen™)

The ApoTarget™ Quick Apoptotic DNA Ladder Detection Kit provides a simple and rapid procedure for extraction of chromosomal DNA. The procedure prepares DNA for use in the methods that detect DNA fragmentation in apoptotic cells. Unlike other kits requiring 1 to 2 days to finish, this detection method requires only less than 90 minutes to prepare DNA in a single tube without the need for extraction or column steps. DNA fragmentation can be easily visualized by agarose gel electrophoresis. This procedure increases recovery of small fragmented DNA, thereby improving the sensitivity of the assay.

Zymosan A (S. cerevisiae) BioParticles™, Alexa Fluor™ 488 conjugate (Invitrogen™)

The Molecular Probes® BioParticles® product line consists of a series of fluorescently labeled, heat- or chemically killed bacteria and yeast in a variety of sizes, shapes, and natural antigenicities. These fluorescent BioParticles® products have been employed to study phagocytosis by fluorescence microscopy, quantitative spectrofluorometry, and flow cytometry.

We offer E. coli (K-12 strain), S. aureus (Wood strain, without protein A) and zymosan (S. cerevisiae) BioParticles® conjugates covalently labeled with a variety of different fluorophores (special care has been taken to remove free dye after conjugation). Unlike the fluorescence of fluorescein-labeled BioParticles® conjugates, which is partially quenched in acidic environments, the fluorescence of the Alexa Fluor®, BODIPY® FL, tetramethylrhodamine and Texas Red® dye conjugates is uniformly intense over the pH range from 3 to 10.

BioParticles Specifications:
• Label (Ex/Em): Alexa Fluor® 488 (~495/519 nm)
• Particle: Zymosan (S. cerevisiae)
Opsonizing reagent available


Using BioParticles Products
BioParticles® conjugates are provided as lyophilized powders. There are approximately 3 x 108 E. coli or S. aureus particles per mg solid and approximately 2 x 107 zymosan particles per mg solid. BioParticles® conjugates can be reconstituted in the buffer of your choice for use in phagocytosis assays. The fluorescence of BioParticles® conjugates that are bound to the surface of the cell (but not internalized) can be quenched by ethidium bromide, trypan blue, or other quenchers. In addition to cellular applications, fluorescent BioParticles® conjugates may be effective as flow cytometry calibration references when sorting bacteria and yeast mutants. These small particles may also be useful references for light scattering studies because their sizes and shapes differ in characteristic ways.

Find More BioParticles® Products
We offer a large range of dye-labeled and unlabeled E. coli (K-12 strain), S. aureus (Wood strain, without protein A), and zymosan (S. cerevisiae) BioParticles® products. Find out about these products and their applications by reviewing Probes for Following Receptor Binding and Phagocytosis—Section 16.1 in the Molecular Probes® Handbook.

For pH-sensitive endocytosis assays, see our pHrodo™ BioParticles® conjugates.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

CaspGLOW™ Fluorescein Active Caspase-9 Staining Kit (Invitrogen™)

The CaspGLOW™ Fluorescein Active Caspase-9 Staining Kit contains all the reagents necessary to detect active caspase-9 in mammalian cells with high sensitivity. Fluorescein (FITC)-conjugated LEHD-FMK, a specific inhibitor of caspase-9, is utilized in this assay for detection. This reagent is cell permeable, non-toxic and irreversibly binds to the active enzyme. Detection of the labeled cells can be determined by flow cytometry, fluorescent microscopy or a fluorescent plate reader.

The caspases constitute a family of aspartate-specific cysteine proteases that mediate a sequence of cleavage events. Recruitment of the inactive proenzyme to oligomerized receptors leads to caspase activation and autoproteolytic cleavage. These active enzymes can then cleave other caspases, thereby generating a caspase signaling cascade that leads to a form of programmed cell death termed apoptosis.

Caspase-9, which is also known as ICE-LAP6 and Mch6, cleaves its substrates at the C-terminal aspartic acid residue of the motif Asp-X-X-Asp. Activation of this signaling protease occurs upon stimulation of death receptors such as CD95/APO-1/Fas, TRAIL-R1, TRAIL-R2, TNFR1, and TRAMP. Caspase-9 is activated by recruitment and dimerization within the Apaf-1 apoptosome. Once recruited, caspase-9 undergoes proteolytic cleavage at Asp315 to yield 35-kDa and 10-kDa fragments. Unlike other caspases, this cleavage event is not required for the catalytic activity of caspase-9. As an initiator caspase, this protease initiates a caspase signaling cascade that results in apoptosis. Substrates of caspase-9 include caspases-3, -6, and -7. Finally, dimerization of cleaved caspase-9 is inhibited by X-linked inhibitor of apoptosis (XIAP).

Reported Application
Flow Cytometric Analysis, Immunocytochemistry

CyQUANT™ LDH Cytotoxicity Assay, fluorescence (Invitrogen™)

The CyQUANT LDH Cytotoxicity Assay, fluorescence, is a fluorescent assay that provides a simple and reliable method for determining cellular cytotoxicity. Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many different cell types that is released into the cell culture medium upon damage to the plasma membrane. The CyQUANT LDH Cytotoxicity Assay, fluorescence, provides the reagents to accurately and quantitatively measure this extracellular LDH.

The CyQUANT LDH Cytotoxicity Assay, fluorescence, features include:
Convenient—add-mix-read assay protocol for adherent and suspension cells, including 3D cell models
Accurate—provides a quantitative measurement of LDH release
Flexible—ideal for high-throughput screening, monitor cytotoxicity from the same sample over time
Robust—formulation with highly purified resazurin results in a large assay dynamic range

LDH is a cytosolic enzyme present in many different cell types and is a well-established and reliable indicator of cellular toxicity. Damage of the plasma membrane results in a release of LDH into the surrounding cell culture medium. This extracellular LDH can be quantified by a coupled enzymatic reaction in which LDH catalyzes the conversion of lactate to pyruvate via NAD+ reduction to NADH. Diaphorase then uses NADH to reduce resazurin to resorufin that can be detected using an excitation of 560 nm and emission of 590 nm. The level of resorufin formation is directly proportional to the amount of LDH released into the medium.

As a consequence of the synthesis and manufacturing processes, all resazurin-based reagents contain a detectable amount of resorufin contamination. The amount of contamination can vary greatly between sources of the material and manufacturing conditions, contributing to differences in detectable background fluorescence. More importantly, the contaminating resorufin reduces the signal to background ratio and dynamic range of the assay. An innovative process was developed that removes the contaminating resorufin, resulting in the highly pure resazurin used in the CyQUANT™ LDH Cytotoxicity Assay, fluorescence.

The CyQUANT LDH Cytotoxicity Assay, fluorescence, provides the reagents needed for the simple, reliable fluorescence-based quantification of cellular cytotoxicity. The kit can be used with different cell types, including 3D cell models, to measure cytotoxicity mediated by chemical compounds as well as cell-mediated cytotoxicity. Since LDH in the medium is the indicator of cellular cytotoxicity, the assay can be used to monitor cytotoxicity from the same sample over time. To perform the assay, an aliquot of the cell culture medium is transferred to a new plate and the reaction mixture is added. After a 10-minute incubation, the reaction is stopped by adding Stop Solution and fluorescence is measured using a microplate reader.

pHrodo™ Green S. aureus BioParticles™ Phagocytosis Kit for Flow Cytometry (Invitrogen™)

The pHrodo™ Green S. aureus BioParticles™ Phagocytosis Kit for Flow Cytometry enables the detection of phagocytic activity in whole blood samples and live cell lines by flow cytometry. The kit includes all the reagents required for assessing particle ingestion and red blood cell lysis. Sufficient reagents are provided in the kit for performing approximately 100 assays using 100 µL of whole blood per assay.

View a selection guide for all pHrodo Indicators used in imaging, flow cytometry and microplate assays.

The pHrodo™ Green S. aureus BioParticles™ assay provides sensitive detection without the need for quenching reagents and extra wash steps, helping save time and minimize the uncertainty of whether particle signals derive from internalized particles.

The pHrodo™ Green S. aureus BioParticles™ Phagocytosis Kit is:

• Specific—fluorogenic, pH-sensitive dye for detection of phagocytosis
• Rapid and reproducible—no wash steps or quencher dye required
• Flexible—multiplex with reagents excited by other laser lines
• Compatible—multiplex with standard reagents and CD markers

How It works
The pHrodo™ Green S. aureus BioParticles™ conjugates are inactivated, unopsonized S. aureus which function as highly sensitive, fluorogenic particles for the detection of phagocytic ingestion. The unique pHrodo™ dye-based system measures phagocytic activity based on acidification of particles as they are ingested, eliminating the wash and quenching steps that are necessary with nonfluorogenic indicators of bacterial uptake.

The pHrodo™ Green Dye
The new Molecular Probes™ proprietary, pH-sensitive, pHrodo™ Green dye is non-fluorescent at neutral pH, but turns bright green upon acidification. Because it is both fluorogenic and pH-sensitive, the pHrodo™ Green dye can be used as a specific sensor of phagocytic events; acidification of the phagosome following phagocytosis is marked by green fluorescence. It is therefore an ideal tool with which to study phagocytosis and its regulation by drugs and/or environmental factors.

The fluorophore is readily excited with the 488 nm argon-ion laser and fluorescence emission can be collected using a standard 530/30 nm BP filter on most flow cytometers, and thus is easily multiplexable with reagents used on other lasers .

Rapid and reproducible
Wash steps and quencher dyes are not needed, since the pHrodo™ Green dye is non-fluorescent outside the cell, making the staining protocol simple and fast. The elimination of wash steps and quencher dyes also improves assay reproducibility, particularly in plate reader-based assays.

Use with bacteria
Analysis of phagocytosis by Gram-positive or Gram-negative bacteria is easily accomplished using the ready-made pHrodo™ Green BioParticles™ conjugates. Use ready-made pHrodo™ Green BioParticles™ conjugates for convenient analysis of phagocytosis of Gram-positive or Gram-negative bacteria, or the amine-reactive pHrodo™ Green STP ester for labeling microorganisms or proteins of your choice.

pHrodo™ Indicators for Phagocytosis
The pHrodo™ Green S. aureus BioParticles™ Phagocytosis Kit is part of a larger product offering of reagents for measurement of phagocytosis. Scientists can use a variety of pHrodo™ reagents in plate readers, fluorescence microscopy imaging and flow cytometry.

Additional information about pHrodo™ Green products, pHrodo™ Red products, and other pHrodo™ indicators for phagocytosis.

LIVE/DEAD™ Fixable Red Dead Cell Stain Kit, for 488 nm excitation (Invitrogen™)

The LIVE/DEAD™ Fixable Red Dead Cell Stain Kit is used to determine the viability of cells prior to the fixation and permeabilization required for intracellular antibody staining or prior to elimination of biohazardous materials using formaldehyde fixation. This kit has been optimized and validated for use with a blue laser flow cytometer.

• Stable—dyes are freeze dried in separate vials to maintain stability

• Robust—staining pattern is the same before and after fixation

• Bright signal—allows for easy distinction between live/dead cells in single channel

View a selection guide for all fixable viability dyes for flow cytometry.

Stable
Unlike products that are sold in solution, the LIVE/DEAD™ Fixable Red Stain has been conveniently packaged in 40-test vials to help ensure the stability and performance of the dye over time. Amine reactive dyes in solution will lose their effectiveness over a short period of time, therefore it is recommended to completely use the vial once rehydrated. If this is not possible, aliquot the vials in small volumes and store at -80°C, avoiding freeze-thaw cycles.

Robust
Dead cell discriminator stains can lose sensitivity after treatment with fixatives such as formaldehyde or ethanol-based fixation methods required for intracellular phosphorylation studies. The LIVE/DEAD™ Fixable Red Stain is an amine reactive dye that binds covalently to intracellular and extracellular amines, and the staining pattern is preserved following formaldehyde fixation.

Optimal brightness
The LIVE/DEAD™ Fixable Red stain was selected based on its fluorescent properties to provide a bright signal when excited with a yellow, green, or blue 488 nm laser. The red-fluorescent reactive dye has an excitation maximum of ~595 nm, making it ideal for use with the yellow laser, and an emission of ~615 nm, allowing collection in the 610 or 630 BP filter. LIVE/DEAD™ Fixable Red Dead Cell stain is also excited well with the blue 488 nm laser. Since live and dead cells can be discriminated using a single dye and a single channel of a flow cytometer, it is an ideal choice for multi-color experiments.

How it works
In cells with compromised membranes, the dye reacts with free amines both in the cell interior and on the cell surface, yielding intense fluorescent staining. In viable cells, the dye's reactivity is restricted to the cell-surface amines, resulting in less intense fluorescence. The difference in intensity is typically greater than 50-fold between live and dead cells, allowing for easy discrimination.

Colors available
LIVE/DEAD™ Fixable Dead Cell Stains are available in a wide variety of colors to meet your multi-color panel needs.

Zymosan A (S. cerevisiae) BioParticles™, Alexa Fluor™ 594 conjugate (Invitrogen™)

The Molecular Probes® BioParticles® product line consists of a series of fluorescently labeled, heat- or chemically killed bacteria and yeast in a variety of sizes, shapes, and natural antigenicities. These fluorescent BioParticles® products have been employed to study phagocytosis by fluorescence microscopy, quantitative spectrofluorometry, and flow cytometry.

We offer E. coli (K-12 strain), S. aureus (Wood strain, without protein A) and zymosan (S. cerevisiae) BioParticles® conjugates covalently labeled with a variety of different fluorophores (special care has been taken to remove free dye after conjugation). Unlike the fluorescence of fluorescein-labeled BioParticles® conjugates, which is partially quenched in acidic environments, the fluorescence of the Alexa Fluor®, BODIPY® FL, tetramethylrhodamine and Texas Red® dye conjugates is uniformly intense over the pH range from 3 to 10.

BioParticles Specifications:
• Label (Ex/Em): Alexa Fluor® 594 (~590/617 nm)
• Particle: Zymosan (S. cerevisiae)
Opsonizing reagent available


Using BioParticles Products
BioParticles® conjugates are provided as lyophilized powders. There are approximately 3 x 108 E. coli or S. aureus particles per mg solid and approximately 2 x 107 zymosan particles per mg solid. BioParticles® conjugates can be reconstituted in the buffer of your choice for use in phagocytosis assays. The fluorescence of BioParticles® conjugates that are bound to the surface of the cell (but not internalized) can be quenched by ethidium bromide, trypan blue, or other quenchers. In addition to cellular applications, fluorescent BioParticles® conjugates may be effective as flow cytometry calibration references when sorting bacteria and yeast mutants. These small particles may also be useful references for light scattering studies because their sizes and shapes differ in characteristic ways.

Find More BioParticles® Products
We offer a large range of dye-labeled and unlabeled E. coli (K-12 strain), S. aureus (Wood strain, without protein A), and zymosan (S. cerevisiae) BioParticles® products. Find out about these products and their applications by reviewing Probes for Following Receptor Binding and Phagocytosis—Section 16.1 in the Molecular Probes® Handbook.

For pH-sensitive endocytosis assays, see our pHrodo™ BioParticles® conjugates.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

Arcturus™ Paradise™ PLUS 2 Round Kit, amino allyl (Applied Biosystems™)

The ARCTURUS® Paradise® PLUS Reagent System permits unprecedented gene expression analysis of millions of previously inaccessible samples. It is an integrated solution for gene expression studies from formalin-fixed, paraffin-embedded (FFPE) tissues, including reagents for tissue staining, RNA isolation and RNA amplification. Amplify RNA from as little as 5 ng of fixed RNA using methods that have been specially optimized to overcome the challenges of cross-linked templates.

This kit provides reagents for 2-round in-vitro transcription, and aminoallyl aRNA labeling.

Key product features:
• Accelerated discovery process – unlock the tissue archives to access previously inaccessible samples
• Simplified procedure – achieve unprecedented results in six steps
• Streamlined application – integrate seamlessly into current workflow
• Efficient workflow – secure downstream success with the Paradise® QC Kit
• Comprehensive system – benefit from the only complete system for formalin-fixed samples

Unlock the Tissue Archives
Currently, tens of millions of tissue samples with known outcomes are available in tissue archives. The Paradise® PLUS Reagent System enables microarray and qRT-PCR analysis of these previously inaccessible samples, allowing retrospective studies to accelerate discoveries.

Achieve Unprecedented Results in Six Steps
Only six steps are required to take you from sample to results (See Figure 1).

Integrate Seamlessly into Current Workflow
The Paradise® PLUS Reagent System is compatible with all major microarray platforms. Robust and reproducible results are achieved using tools and techniques commonly employed in today’s research laboratories (See Figures 2, 3, 4, and 5).

Secure Downstream Success with the Paradise® QC Kit
Not all FFPE samples contain high quality RNA. Identify the best samples for your studies using a simple, real-time PCR research technique, and then proceed with confidence using these pre-qualified samples (See Figure 6).

Benefit from the Only Complete System for Formalin-fixed Samples
The Paradise® PLUS Reagent System is the only available solution that provides all the components necessary to prepare RNA from formalin-fixed, paraffin-embedded tissue samples for gene expression analysis. To ensure successful microarray results, the system includes reagents optimized for exceptional recovery of RNA and superior RNA amplification.

Integrated Systems for Microgenomics
The Paradise PLUS Reagent System is a validated as part of the ARCTURUS® Complete System for Microgenomics®, an integrated solution for utilizing small quantities of RNA for gene expression analysis. This system features the ARCTURUSXT™ Laser Capture Microdissection (LCM) Instrument to procure pure cell populations. ACTURUS® Systems for Microgenomics enable accurate and sensitive microarray assays that reveal molecular signatures otherwise obscured in whole tissue samples or cell mixtures.

For Research Use Only. Not for use in diagnostics procedures.

Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & Propidium Iodide (PI) (Invitrogen™)

The Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & Propidium Iodide is a flow cytometry kit used to measure early apoptosis by detecting phosphatidyl serine expression and membrane permeability.

View a selection guide for all apoptosis assays for flow cytometry.

Superior brightness
Unlike other Annexin V kits that have lower protein concentrations or purification levels, the Annexin V Alexa Fluor™ 488 conjugate is optimized for flow cytometry to provide the largest separation between apoptotic and live cells. The Alexa Fluor™ 488 dye is a superior green-fluorescent dye with a spectrum similar to fluorescein (FITC).

High binding efficiency
Annexin V conjugates are made from a highly purified cys-annexin, which leads to higher binding efficiency, resulting in highly accurate characterization of the apoptotic process.

Multi-parametric
Many publications require a minimum of two different ways to identify that cells are apoptotic. This multi-parametric kit detects phosphatidyl serine (PS) on the cytoplasmic surface of the cell membrane and membrane integrity using propidium iodide.

How it works
When cells are stained with Annexin V and propidium iodide, apoptotic cells expressing PS show green fluorescence, which can be detected in the FITC channel, and low red fluorescence. Dead or necrotic cells show bright red fluorescence and no green fluorescence, while live cells show no green or red fluorescence.

Pacific Blue™ Annexin V/SYTOX™ AADvanced™ Apoptosis Kit, for flow cytometry (Invitrogen™)

This product detects the externalization of phosphatidylserine in apoptotic cells using recombinant annexin V conjugated to violet-fluorescent Pacific Blue™ dye and dead cells using SYTOX™ AADvanced™ stain. After staining a cell population with Pacific Blue™, annexin V and SYTOX™ AADvanced™, apoptotic cells show violet fluorescence, dead cells show red fluorescence, and live cells show little or no fluorescence. These populations are easily distinguished by a flow cytometer with the 405 nm and 488 nm lines for excitation. There is very little spectral overlap between the two dyes, therefore very little compensation is needed. Each kit contains sufficient reagents for approximately 50 flow cytometry tests.

View a selection guide for all apoptosis assays for flow cytometry.

Amplex™ Red Glucose/Glucose Oxidase Assay Kit (Invitrogen™)

The Amplex® Red Glucose/Glucose Oxidase Assay Kit provides a sensitive and simple method for detecting d-glucose or glucose oxidase using a fluorescence microplate reader or fluorometer.

See our complete line of Fluorescence Microplate assays.

• Detect concentrations as low as 3 µM d-glucose or 0.05 mU/mL of glucose oxidase
• Format allows for multiple time point measurements
• Designed for minimal autofluorescence interference

The Amplex® Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) is a colorless, stable, and extremely versatile peroxidase substrate. Because peroxidase- and glucose oxidase-mediated reactions can be coupled, it is possible to measure glucose oxidase activity or the release of glucose by any glucosidase enzyme—for instance, β-glucosidase and glucocerebrosidase—in either a continuous or discontinuous assay. Because of the ability to couple reactions, this assay has been demonstrated to be useful for the quantification of glucose levels in foods, fermentation media, and bodily fluids.

In the assay, glucose oxidase reacts with d-glucose to form d-gluconolactone and hydrogen peroxide. In the presence of horseradish peroxidase (HRP), hydrogen peroxide then reacts with the Amplex® Red reagent in a 1:1 stoichiometric ratio to generate the red-fluorescent product resorufin.

Because resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, there is little interference from autofluorescence in most biological samples.

Use Amplex® Red Assays for a Broad Range of Investigations
A wide variety of validated Amplex® Red assays are available for studying cell signaling and lipids, neurobiology, inflammation and immune function, and metabolism. We also offer Amplex® UltraRed Reagent (Cat. No. A36006), a second-generation reagent providing greater sensitivity and brighter fluorescence, and the Amplex® Red/UltraRed Stop Reagent (Cat. No. A33855). The Amplex® Red/UltraRed Stop Reagent provides convenience and control by allowing the fluorescence signal-generating reaction to be terminated at a user-determined time point. After addition of the stop reagent, the fluorescence signal remains stable for at least three hours. Custom assay design and packaging are also available.

CellTrace™ Yellow Cell Proliferation Kit, for flow cytometry (Invitrogen™)

CellTrace™ Yellow Cell Proliferation Kit is used for in vitro and in vivo labeling of cells to trace multiple generations using dye dilution by flow cytometry. This reagent is specifically designed for excitation by either a 532-nm or 561-nm laser, and it can be easily multiplexed with reagents and antibodies excited by other commonly used lasers.

Superior performance—bright, single-peak staining enables visualization of multiple generations
Long-term signal stability—well retained in cells for several days post stain
Versatile—multiple colors available to easily combine with antibodies or markers of cell function
Simple—robust staining protocol

Additional information is available about other CellTrace™ Reagents to meet your multiplexing needs.

Superior fluorescent staining
Successful proliferation analysis by dye dilution requires an extremely bright dye to distinguish fluorescently labeled cells from auto-fluorescence after several cell divisions. The intense fluorescent staining provided by CellTrace Yellow dye enables the visualization of six or more generations of proliferating cells before the signal is overwhelmed by intrinsic cellular auto-fluorescence. This kit enables consistent, homogeneous staining results with little fluorescence variation between cells in a population, so distinct generations can be seen without any requirement for complex modeling software.

Long-term signal retention
Unlike stains that label the lipid membrane of cells, CellTrace Yellow stain easily crosses the plasma membrane and covalently binds inside cells where the stable, well-retained fluorescent dye offers a consistent signal, even after several days in a cell culture environment. CellTrace Yellow dye binds covalently to all free amines on the surface and inside of cells and shows little cytotoxicity, with minimal observed effect on the proliferative ability or biology of cells.

Easy multiplexing with other fluorophores
The yellow excitation at 546 nm and emission at 579 nm of CellTrace Yellow dye make it ideal for multiplexing due to the limited spectral overlap with other common dyes (e.g., Alexa Fluor™488 and FITC) and green fluorescent protein (GFP) .

Use the SpectraViewer to help you plan your experiments.

Simple, robust staining protocol
The CellTrace Yellow Cell Proliferation Kit contains convenient single-use vials of dry dye to permit small-scale experiments without preparing excess quantities of dye. A stock solution is prepared by dissolving the contents of a vial in anhydrous DMSO prior to use. To stain 1 mL of cells in protein-free medium, 1 µL of this stock solution is typically used. Cells should be stained for 20 minutes at room temperature with gentle agitation. A brief wash with complete medium will then quench any dye remaining in solution.

Related links
General information about Thermo Fisher Scientific flow cytometry products and support is also available.

ReadyProbes™ Cell Viability Imaging Kit, Blue/Green (Invitrogen™)

ReadyProbes® Cell Viability Imaging Kit (Blue/Green) is a ready-to-use assay that can quickly and easily determine the viability of cells. Just add 2 drops each of room temperature, stable NucBlue® Live reagent (Hoechst 33342) and NucGreen® Dead reagent to 1 mL of cell growth media, then determine viability by counting total vs dead cells. NucBlue® Live reagent stains the nuclei of all the cells and can be detected with a standard DAPI filter. NucGreen® Dead reagent stains only the nuclei of cells with compromised plasma membrane integrity and is detected using a standard FITC/GFP (green) filter set. This kit is appropriate for fluorescence microscopy, fluorescence microplate readers, and flow cytometry.

NucBlue® Live reagent: stains the nuclei of all cells; detected with a standard DAPI filter (excitation/emission maxima: 360/460 nm)
NucGreen® Dead reagent: stains only the nuclei of dead cells with compromised plasma membranes; detected with standard FITC/GFP (green) filter set (excitation/emission maxima: 504/523 nm)
See other ReadyProbes® reagents for cell staining
Learn more about other assays for cell viability

Suggestions for use
• NucBlue® Live and NucGreen® Dead reagents may be added directly to cells in full growth media or a compatible buffer solution.
• In most cases, 2 drops/mL and an incubation time of 5 to 30 minutes will give bright nuclear staining; however, optimization may be needed for some cell types, conditions, and applications. In such cases, simply add more or fewer drops until the optimal staining intensity is obtained.

Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 594 dye (Invitrogen™)

The Click-iT EdU Cell Proliferation Kit for Imaging is a superior alternative to traditional proliferation assays that is optimized for fluorescence microscopy applications. In this assay the modified thymidine analogue EdU is efficiently incorporated into newly synthesized DNA and fluorescently labeled with a bright, photostable Alexa Fluor® dye in a fast, highly-specific click reaction. This fluorescent labeling of proliferating cells is accurate and compatible with antibody methods due to the mild click protocol.

• Simple—works the first time, every time, in less time
• Efficient—no denaturation steps or harsh treatment required
• Content-rich results—better preservation of cell morphology, antigen structure, and DNA integrity
• Consistent—not dependent on variable antibody lots for detection

The most accurate proliferation-detection methods are based on the incorporation and measurement of nucleoside analogues in newly synthesized DNA, with bromodeoxyuridine (BrdU) a commonly used analogue. BrdU-labeled DNA is quantitated using anti-BrdU antibodies following DNA denaturation by harsh methods (HCl, heat, or enzymes) to expose the BrdU molecules. This step is time consuming and difficult to perform consistently. The harsh treatment can also adversely effect sample integrity and quality, which makes co-staining with other antibodies challenging.

Superior Proliferation Methodology
The Click-iT EdU Cell Proliferation Kit for Imaging provides a superior alternative to BrdU assays for measuring cell proliferation. EdU (5-ethynyl-2'-deoxyuridine) is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis. With Click-iT® EdU, mild fixation and detergent permeabilization is sufficient for the small molecule-based Click-iT® EdU detection reagent to gain access to the DNA. As a consequence, the Click-iT® EdU imaging kit is not only easy to use, but more accurate and compatible with cell cycle analysis and other intracellular or extracellular targets for truly content-rich results.

This kit is optimized for fluorescence microscopy applications; visit the Click-iT® technology area of our website for kits designed for high-content imaging, fluorescence microplate, or flow cytometry platforms.

Learn more about Click-iT technology >
Find more tools for image-based detection of proliferating cells >

Notes:
The Click-iT® assay can be used on cells in culture or in vivo following EdU administration by feeding or injection methods.
The Click-iT® assay can be used with BrdU in dual pulse experiments by using the anti-BrdU (clone MoBu-1) antibody, which does not cross react with EdU.
The Click-iT® technology is compatible with immunohistochemical, immunocytochemical, and fluorescent dyes that are fixation tolerant or designed for fixed cell labeling.

LIVE/DEAD™ Fixable Far Red Dead Cell Stain Kit, for 633 or 635 nm excitation (Invitrogen™)

The LIVE/DEAD™ Fixable Far-Red Dead Cell Stain Kit is used to determine the viability of cells prior to the fixation and permeabilization required for intracellular antibody staining or prior to elimination of biohazardous materials using formaldehyde fixation. This kit has been optimized and validated for use with a red laser flow cytometer.

• Stable—dyes are freeze dried in separate vials to maintain stability

• Robust—staining pattern is the same before and after fixation

• Bright signal—allows for easy distinction between live/dead cells in single channel

View a selection guide for all fixable viability dyes for flow cytometry.

Stable
Unlike products that are sold in solution, the LIVE/DEAD™ Fixable Far-Red Stain has been conveniently packaged in 40-test vials to help ensure the stability and performance of the dye over time. Amine reactive dyes in solution will lose their effectiveness over a short period of time, therefore it is recommended to completely use the vial once rehydrated. If this is not possible, aliquot the vials in small volumes and store at -80°C, avoiding freeze-thaw cycles.

Robust
Dead cell discriminator stains can lose sensitivity after treatment with fixatives such as formaldehyde or ethanol-based fixation methods required for intracellular phosphorylation studies. The LIVE/DEAD™ Fixable Far-Red Stain is an amine reactive dye that binds covalently to intracellular and extracellular amines, and the staining pattern is preserved following formaldehyde fixation.

Optimal brightness
The LIVE/DEAD™ Fixable Far-Red Stain was selected based on its fluorescent properties to provide a bright signal when excited with a red laser. The far-red fluorescent reactive dye has an excitation maximum of ~633 nm making it ideal for use with the red or HeNe laser with an emission of ~655 nm. Since live and dead cells can be discriminated using a single dye and a single channel of a flow cytometer, it is an ideal choice for multi-color experiments.

How it works
In cells with compromised membranes, the dye reacts with free amines both in the cell interior and on the cell surface, yielding intense fluorescent staining. In viable cells, the dye's reactivity is restricted to the cell-surface amines, resulting in less intense fluorescence. The difference in intensity is typically greater than 50-fold between live and dead cells, allowing for easy discrimination.

Colors available
LIVE/DEAD™ Fixable Dead Cell Stains are available in a wide variety of colors to meet your multi-color panel needs.

pHrodo™ Red Zymosan Bioparticles™ Conjugate for Phagocytosis (Invitrogen™)

New pH-sensitive pHrodo® Red dye conjugates give faster and more accurate results than any other phagocytosis assay. pHrodo® Red dye conjugates are non-fluorescent outside the cell, but fluoresce brightly red in phagosomes. Get faster staining and more accurate results—without the need for wash steps or quencher dye.

• Specific detection of phagocytosis and endocytosis
• Reduced signal variability and improved timing in sensitive experiments
• Multiplex with green dyes such as GFP, Fluo-4, or calcein

The fluorescence of the novel pHrodo® Red dye dramatically increases as pH decreases from neutral to acidic, making it an ideal tool to study phagocytosis and its regulation by drugs and/or environmental factors. The lack of fluorescence outside the cell eliminates the need for wash steps and quencher dyes.

Use the ready-made pHrodo® Red Zymosan BioParticles® Conjugate in imaging or flow applications, or pHrodo® Red SE, the activated succinimidyl ester, for labeling microorganisms or proteins of your choice.

In addition, pHrodo® dye is also available in green color, with E. coli, zymosan, S. aureus, and dextran 10,000 conjugates.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

LIVE/DEAD™ Cell Imaging Kit (488/570) (Invitrogen™)

The LIVE/DEAD® Cell Imaging Kit is a sensitive two-color fluorescence cell viability assay optimized for FITC and Texas Red® filters. Quick and easy to use, the kit allows discrimination between live and dead cells with two probes that measure recognized parameters of cytotoxicity and cell viability—intracellular esterase activity and plasma membrane integrity.

The LIVE/DEAD® Cell Imaging Kit *488/570* offers:

Fast, simple determination of live and dead cells

• Accuracy with convenience
• Sensitive probes ideal for FITC and Texas Red filters


Dual Probe-Based Assay for Imaging Platforms
Just like our popular LIVE/DEAD® Viability/Cytotoxicity assay, the new LIVE/DEAD® Cell Imaging Kit is based on a cell-permeable dye for staining of live cells and a cell-impermeable dye for staining of dead and dying cells, which are characterized by compromised cell membranes. To adapt this important assay for imaging platforms, the LIVE/DEAD® Cell Imaging Kit components were optimized for the common green and red imaging filters used with FITC and Texas Red. The live cell component produces an intense, uniform green fluorescence in live cells (ex/em 488 nm/515 nm). The dead cell component produces a predominantly nuclear red fluorescence (ex/em 570nm/602 nm) in cells with compromised cell membranes, a strong indicator of cell death and cytotoxicity (Fig 1).

Quick, Exact Determination of Viability and Cytotoxicity
The LIVE/DEAD® Cell Imaging kit provides very sensitive detection of key measures of cell health: viability, live/dead ratio, and cytotoxicity. The components of the LIVE/DEAD® Cell Imaging kit are configured for ease of use with minimal dilutions.

Assay Principle
With the LIVE/DEAD® Cell Imaging Kit, live cells are distinguished by the presence of ubiquitous intracellular esterase activity as determined by the enzymatic conversion of the virtually non-fluorescent cell-permeant calcein AM to the intensely fluorescent calcein, which is well-retained within live cells. The red component of the LIVE/DEAD® Cell Imaging Kit is cell-impermeant and therefore only enters cells with damaged membranes. In dying and dead cells a bright red fluorescence is generated upon binding to DNA. Background fluorescence levels are inherently low with this assay technique because the dyes are virtually non-fluorescent before interacting with cells. The fluorophores in the LIVE/DEAD® Cell Imaging Kit were selected for their brightness, spectral properties (FITC and Texas Red filters), and ease of use. Packaged for workflow convenience, they allow for effortless and quick determination of the fractions of live and dead cells in a population.

See other Molecular Probes imaging tools and reagents

eBioscience™ Annexin V-Biotin Apoptosis Detection Kit (Invitrogen™)

Annexins are a family of calcium-dependent phospholipid-binding proteins, which bind to phosphatidylserine (PS) to identify apoptotic cells. In healthy cells, PS is predominantly located along the cytosolic side of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution in the phospholipid bilayer and translocates to the extracellular membrane, which is detectable with fluorescently labeled Annexin V. In early stages of apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI) and 7-AAD, therefore cells which display only Annexin V staining (PI/7-AAD negative) are in early stages of apoptosis. During late-stage apoptosis, loss of cell membrane integrity allows Annexin V binding to cytosolic PS, as well as cell uptake of PI and 7-AAD. Annexin V staining, paired with 7-AAD or PI is widely used to identify apoptotic stages by flow cytometry.

Host
E.coli

Conjugate
Biotin

Purity
> 98% pure

Molecular Mass
35.8 kDa

Reported Application
Flow Cytometric Analysis

eBioscience™ Essential Human Treg Phenotyping Panel & Staining Buffers (Invitrogen™)

The Invitrogen eBioscience Essential Human Treg Phenotyping Panel & Staining Buffers is designed for flow cytometry-based identity testing of regulatory T-cells (Tregs). It leverages high quality eBioscience antibodies and fixation/permeabilization reagents (for the intracellular marker FOXP3) and includes a ready-to-use protocol for experimental set-up, gating strategy, and analysis.

Benefits include:
Proven performance―antibodies and supporting reagents designed for flow cytometry
Highly referenced antibodies―includes one of the most referenced clones for FOXP3
Turnkey kit and protocol―contains reagents and standardized gating recommendations needed to perform flow cytometry characterization

eBioscience™ Annexin V Apoptosis Detection Kit APC (Invitrogen™)

Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.

In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD, or Fixable Viability Dyes such as eFluor™ 660 or eFluor™ 780. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).

Note: Fixable Viability Dye eFluor™ 450 is not recommended for use with Annexin V Apoptosis Detection Kits.

Reported Application
Flow Cytometric Analysis

Click-iT™ EdU Imaging Kit with Alexa Fluor™ 488, 594, and 647 Azides (Invitrogen™)

The Click-iT® EdU Imaging Kit is a trial version of our superior alternative to traditional proliferation assays optimized for fluorescence microscopy applications. In this assay the modified thymidine analogue EdU is efficiently incorporated into newly synthesized DNA and fluorescently labeled with a bright, photostable Alexa Fluor® dye in a fast, highly-specific click reaction. This fluorescent labeling of proliferating cells is accurate and compatible with antibody methods due to the mild click protocol. This trial-size kit provides sufficient EdU to label up to 6–30 coverslips and enough Alexa Fluor® 488, 594, and 647 azide to detect new DNA synthesis on 2–10 coverslips with each fluorophore.
• Simple—works the first time, every time, in less time
• Efficient—no denaturation steps or harsh treatment required
• Content-rich results—better preservation of cell morphology, antigen structure, and DNA integrity
• Consistent—not dependent on variable antibody lots for detection

The most accurate proliferation-detection methods are based on the incorporation and measurement of nucleoside analogues in newly synthesized DNA, with bromodeoxyuridine (BrdU) a commonly used analogue. BrdU-labeled DNA is quantitated using anti-BrdU antibodies following DNA denaturation by harsh methods (HCl, heat, or enzymes) to expose the BrdU molecules. This step is time consuming and difficult to perform consistently. The harsh treatment can also adversely effect sample integrity and quality, which makes co-staining with other antibodies challenging.

Superior Proliferation Methodology
The Click-iT® EdU Imaging Kit provides a superior alternative to BrdU assays for measuring cell proliferation. EdU (5-ethynyl-2'-deoxyuridine) is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis. With Click-iT® EdU, mild fixation and detergent permeabilization is sufficient for the small molecule-based Click-iT® EdU detection reagent to gain access to the DNA. As a consequence, the Click-iT® EdU Imaging Kit is not only easy to use, but more accurate and compatible with cell cycle analysis and other intracellular or extracellular targets for truly content-rich results.

This kit is optimized for fluorescence microscopy applications; visit the Click-iT® technology area of our website for kits designed for high-content imaging, fluorescence microplate, or flow cytometry platforms.

Learn more about Click-iT® technology

Notes:
The Click-iT® assay can be used on cells in culture or in vivo following EdU administration by feeding or injection methods.
The Click-iT® assay can be used with BrdU in dual pulse experiments by using the anti-BrdU (clone MoBu-1) antibody, which does not cross react with EdU.
The Click-iT® technology is compatible with immunohistochemical, immunocytochemical, and fluorescent dyes that are fixation tolerant or designed for fixed cell labeling.

Dead Cell Apoptosis Kit with Annexin V FITC and PI, for flow cytometry (Invitrogen™)

This product detects the externalization of phosphatidylserine in apoptotic cells using recombinant annexin V conjugated to green-fluorescent FITC dye and dead cells using propidium iodide (PI). Propidium iodide stains necrotic cells with red fluorescence. After treatment with both probes, apoptotic cells show green fluorescence, dead cells show red and green fluorescence, and live cells show little or no fluorescence.

View a selection guide for all apoptosis assays for flow cytometry.

Click-iT™ Plus EdU Alexa Fluor™ 350 Flow Cytometry Assay Kit (Invitrogen™)

The Click-iT Plus EdU Alexa Fluor 350 Flow Cytometry Assay Kit provides a simplified, more robust assay for analyzing DNA replication in proliferating cells compared to traditional BrdU methods. Newly synthesized DNA is analyzed using the UV laser of the flow cytometer. The Click-iT Plus formulation is compatible with standard fluorophores, including R-PE and R-PE tandems, as well as fluorescent proteins.

• Multiplexable—compatible with R-PE (and tandems) and fluorescent proteins
• Accurate—superior results compared to BrdU assays
• Fast—results in as little as 60 minutes

View selection guide for all Click-iT EdU and Click-iT Plus EdU assays for flow cytometry.

Multiplexable
Click-iT Plus Alexa Fluor 350 EdU assays can be used in conjunction with R-PE and R-PE tandems, as well as fluorescent proteins such as GFP and mCherry. The Alexa Fluor 350 labeling reagent is excited readily at 350 nm with a UV laser and emits at 438 nm.

An Advanced Method Giving You Results Superior to BrdU
The most accurate method of proliferation analysis is direct measurement of DNA synthesis. Originally, this was performed through incorporation of radioactive nucleosides. This method was replaced by antibody-based detection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT Plus EdU Flow Cytometry assay is a novel alternative to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog that is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry, which is a copper-catalyzed covalent reaction between an azide and an alkyne. In this application, the alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to the Alexa Fluor dye. Standard flow cytometry methods are used for determining the percentage of S-phase cells in the population.

Mild Conditions Allow Use with Cell Cycle Dyes and Antibodies
The small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions, while standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT Plus detection reagent to gain access to the DNA. This is in contrast to BrdU assays that require DNA denaturation (using HCl, heat, or digestion with DNase) to expose the BrdU so that it can be detected with an anti-BrdU antibody. Sample processing for the BrdU assay can result in signal alteration of the cell cycle distribution, as well as destruction of antigen recognition sites when using the HCl method. In contrast, the easy-to-use Click-iT Plus EdU assay is compatible with cell cycle dyes. The Click-iT Plus EdU assay can also be multiplexed with antibodies against surface and intracellular markers, as well as conjugates labeled with standard fluorophores including R-PE, R-PE tandems, and fluorescent proteins (GFP and mCherry).

Quick and Simple Protocol
The Click-iT Plus EdU protocol is based on the aldehyde fixation and detergent permeabilization steps for immunohistochemical antibody labeling. However, EdU is compatible with other fixation/permeabilization agents including saponin and methanol. In just five steps you’ll be ready to analyze your cell proliferation data:

1. Treat cells with EdU.
2. Fix and permeabilize cells.
3. Detect S-phase cells with Click-iT Plus detection cocktail for 30 min.
4. Wash once.
5. Analyze.

Results can be seen in as little as 60 minutes in some circumstances, but we recommend 90 minutes for all applications.

LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit, for 633 or 635 nm excitation (Invitrogen™)

The LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit is used to determine the viability of cells prior to the fixation and permeabilization required for intracellular antibody staining or prior to elimination of biohazardous materials using formaldehyde fixation. This kit has been optimized and validated for use with a red laser flow cytometer.

• Stable—dyes are freeze dried in separate vials to maintain stability
• Robust—staining pattern is the same before and after fixation
• Bright signal—allows for easy distinction between live/dead cells in single channel

View a selection guide for all fixable viability dyes for flow cytometry.

Stable
Unlike products that are sold in solution, the LIVE/DEAD™ Fixable Near-IR Stain has been conveniently packaged in 40-test vials to help ensure the stability and performance of the dye over time. Amine reactive dyes in solution will lose their effectiveness over a short period of time, therefore it is recommended to completely use the vial once rehydrated. If this is not possible, aliquot the vials in small volumes and store at -80°C, avoiding freeze-thaw cycles.

Robust
Dead cell discriminator stains can lose sensitivity after treatment with fixatives such as formaldehyde or ethanol-based fixation methods required for intracellular phosphorylation studies. The LIVE/DEAD™ Fixable Near-IR Stain is an amine reactive dye that binds covalently to intracellular and extracellular amines, and the staining pattern is preserved following formaldehyde fixation.

Optimal brightness
The LIVE/DEAD™ Fixable Near-IR stain was selected based on its fluorescent properties to provide a bright signal when excited with a red laser. The near-IR fluorescent reactive dye has an excitation maximum of ~633 nm making it ideal for use with the red or HeNe laser with an emission of ~750 nm. Since live and dead cells can be discriminated using a single dye and a single channel of a flow cytometer, it is an ideal choice for multi-color experiments.

How it works
In cells with compromised membranes, the dye reacts with free amines both in the cell interior and on the cell surface, yielding intense fluorescent staining. In viable cells, the dye's reactivity is restricted to the cell-surface amines, resulting in less intense fluorescence. The difference in intensity is typically greater than 50-fold between live and dead cells, allowing for easy discrimination.

Colors available
LIVE/DEAD™ Fixable Dead Cell Stains are available in a wide variety of colors to meet your multi-color panel needs.

Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit, for flow cytometry (Invitrogen™)

The Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit provides a simple and fast method for the detection of apoptosis with dead cell discrimination by flow cytometry. The Violet Ratiometric Membrane Asymmetry Probe, 4'-N,N-diethylamino-6-(N,N,N-dodecyl-methylamino-sulfopropyl)-methyl-3-hydroxyflavone (F2N12S), is a novel violet excitable dye for the detection of membrane asymmetry changes during apoptosis. The dye exhibits an excited-state intramolecular proton transfer (ESIPT) reaction resulting in a dual fluorescence with two emission bands corresponding to 530 nm and 585 nm, producing a two-color ratiometric response to variations in surface charge. Ratiometric probes have several advantages over traditional fluorochrome labeled reagents. The ratiometric probe is a self-calibrating absolute parameter of apoptotic transformation, which is independent of probe concentration, cell size, and instrument variation, such as fluctuations of laser intensity or sensitivity of the detectors. Given that apoptosis modifies the surface charge of the outer leaflet of the plasma membrane the violet membrane asymmetry probe F2N12S can monitor changes in membrane asymmetry that occur during apoptosis through a change in the relative intensity of the two emission bands of the dye. The F2N12S probe is combined with SYTOX(R) AADvanced dead cell stain, which is capable of passing through the cell membrane only in late apoptotic or necrotic cells allowing discrimination form early apoptotic cells. Samples can be analyzed after a 5 minute incubation at room temperature and does not require special buffers or wash steps. This kit can be paired with other reagents such as MitoProbe™ DiIC1(5) or annexin V for multiparametric analysis of apoptosis and viability. This reagent kit allow researchers to maximaize the utility of their instruments by utilizing the violet laser Each kit contains sufficient reagents for ~100 flow cytometry tests.

View a selection guide for all apoptosis assays for flow cytometry.

LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation (Invitrogen™)

The LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit is used to determine the viability of cells prior to the fixation and permeabilization required for intracellular antibody staining or prior to elimination of biohazardous materials using formaldehyde fixation. This kit has been optimized and validated for use with a violet laser flow cytometer.

• Stable—dyes are freeze dried in separate vials to maintain stability

• Robust—staining pattern is the same before and after fixation

• Low compensation—minimal spectral overlap between other fluorophores

View a selection guide for all fixable viability dyes for flow cytometry.

Stable
Unlike products that are sold in solution, the LIVE/DEAD™ Fixable Aqua Stain has been conveniently packaged in 40-test vials to help ensure the stability and performance of the dye over time. Amine reactive dyes in solution will lose their effectiveness over a short period of time, therefore it is recommended to completely use the vial once rehydrated. If this is not possible, aliquot the vials in small volumes and store at -80°C, avoiding freeze-thaw cycles.

Robust
Dead cell discriminator stains can lose sensitivity after treatment with fixatives such as formaldehyde or ethanol-based fixation methods required for intracellular phosphorylation studies. The LIVE/DEAD™ Fixable Aqua Stain is an amine reactive dye that binds covalently to intracellular and extracellular amines, and the staining pattern is preserved following formaldehyde fixation.

Low compensation
The LIVE/DEAD™ Fixable Aqua Stain was selected based on its fluorescent properties to minimize compensation between other violet dyes and dyes that excite off of the 488 nm blue laser. The aqua-fluorescent reactive dye has an excitation maximum of ~375 nm, but it is excited well with the 405 nm violet laser, and it has an emission maxima of ~512 nm, so it can be collected in the second channel on most violet laser flow cytometers.

How it works
In cells with compromised membranes, the dye reacts with free amines both in the cell interior and on the cell surface, yielding intense fluorescent staining. In viable cells, the dye's reactivity is restricted to the cell-surface amines, resulting in less intense fluorescence. The difference in intensity is typically greater than 50-fold between live and dead cells, allowing for easy discrimination.

Colors available
LIVE/DEAD™ Fixable Dead Cell Stains are available in a wide variety of colors to meet your multi-color panel needs.

CellTrace™ Blue Cell Proliferation Kit, for flow cytometry (Invitrogen™)

The CellTrace Blue Cell Proliferation Kit is used for in vitro and in vivo labeling of cells to trace multiple generations using dye dilution by flow cytometry. The CellTrace Blue reagent is specifically designed for excitation by a UV laser (at 355 nm or 375 nm), and it can be easily multiplexed with reagents and antibodies excited by other commonly used lasers.

• Superior performance—bright, single-peak staining enables visualization of multiple generations
• Long-term signal stability—well-retained in cells for several days post stain
• Versatile—multiple colors available to easily combine with antibodies or markers of cell function, such as GFP
• Simple, robust staining protocol

View a selection guide for all CellTrace Cell Proliferation Kits for flow cytometry.

Superior fluorescent staining
Successful proliferation analysis by dye dilution (see figure below) requires an extremely bright dye to distinguish fluorescently labeled cells from auto-fluorescence after several cell divisions. The intense fluorescent staining provided by CellTrace Blue dye enables the visualization of five or more generations of proliferating cells before the signal is overwhelmed by intrinsic cellular auto-fluorescence. Consistent, homogeneous staining results in very little fluorescence variation between cells in a population, so distinct generations can be seen without any requirement for complex modeling software.The recommended working concentration for peripheral blood mononuclear cells is 5–10 µM. However, as with all reagents used in flow cytometry, to achieve optimal signal, reagent titration is highly recommended.

Long-term signal retention
Unlike stains that label the lipid membrane of cells, CellTrace Blue stain easily crosses the plasma membrane and covalently binds inside cells where the stable, well-retained fluorescent dye provides a consistent signal, even after several days in a cell culture environment. CellTrace Blue dye binds covalently to all free amines on the surface and inside of cells and shows little cytotoxicity, with minimal observed effect on the proliferative ability or biology of cells.

Easy multiplexing with other fluorophores
The UV excitation and narrow emission of CellTrace Blue dye make it ideal for multiplexing as it lies outside the spectral emission profiles of most other dyes and fluorescent proteins.

Simple, robust staining protocol
The CellTrace Blue Cell Proliferation Kit contains convenient single-use vials of dry dye to permit small-scale experiments without preparing excess quantities of dye. A stock solution is prepared by dissolving the contents of a vial in anhydrous DMSO prior to use. To stain 1 mL of cells in protein-free medium, 1 µL of this stock solution is typically used. Cells should be stained for 20 minutes at room temperature with gentle agitation. A brief wash with complete medium will then quench any dye remaining in solution.

ReadyProbes™ Cell Viability Imaging Kit, Blue/Red (Invitrogen™)

ReadyProbes® Cell Viability Imaging Kit (Blue/Red) is a ready-to-use kit that can quickly and easily determine the viability of cells. Just add 2 drops each of room temperature, stable NucBlue® Live reagent (Hoechst 33342) and propidium iodide to 1 mL of cell growth media, then determine viability by counting total vs dead cells. NucBlue® Live reagent stains the nuclei of all the cells and can be detected using a standard DAPI filter. Propidium iodide stains only the nuclei of cells with compromised plasma membrane integrity and is detected with a standard TRITC/RFP (orange) filter set. This kit is appropriate for fluorescence microscopy, fluorescence micro plate readers, and flow cytometry.

NucBlue® Live reagent: stains the nuclei of all cells; detected with a standard DAPI filter (excitation/emission maxima: 360/460 nm)
Propidium iodide: stains the nuclei of dead cells with compromised plasma membrane; detected with standard TRITC/RFP (orange) filter set (excitation/emission maxima: 535/617 nm)
See other ReadyProbes® reagents for cell staining
Learn more about other assays for cell viability

Suggestions for use
• NucBlue® Live reagent and propidium iodide can be added directly to cells in full growth media or a compatible buffer solution.
• In most cases, 2 drops/mL and an incubation time of 5 to 30 minutes will give bright nuclear staining; however, optimization may be needed for some cell types, conditions, and applications. In such cases, simply add more or fewer drops until the optimal staining intensity is obtained.

Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 647 dye (Invitrogen™)

The Click-iT EdU Cell Proliferation Kit for Imaging is a superior alternative to traditional proliferation assays that is optimized for fluorescence microscopy applications. In this assay the modified thymidine analogue EdU is efficiently incorporated into newly synthesized DNA and fluorescently labeled with a bright, photostable Alexa Fluor® dye in a fast, highly-specific click reaction. This fluorescent labeling of proliferating cells is accurate and compatible with antibody methods due to the mild click protocol.

• Simple—works the first time, every time, in less time
• Efficient—no denaturation steps or harsh treatment required
• Content-rich results—better preservation of cell morphology, antigen structure, and DNA integrity
• Consistent—not dependent on variable antibody lots for detection

The most accurate proliferation-detection methods are based on the incorporation and measurement of nucleoside analogues in newly synthesized DNA, with bromodeoxyuridine (BrdU) a commonly used analogue. BrdU-labeled DNA is quantitated using anti-BrdU antibodies following DNA denaturation by harsh methods (HCl, heat, or enzymes) to expose the BrdU molecules. This step is time consuming and difficult to perform consistently. The harsh treatment can also adversely effect sample integrity and quality, which makes co-staining with other antibodies challenging.

Superior Proliferation Methodology
The Click-iT EdU Cell Proliferation Kit for Imaging provides a superior alternative to BrdU assays for measuring cell proliferation. EdU (5-ethynyl-2'-deoxyuridine) is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis. With Click-iT® EdU, mild fixation and detergent permeabilization is sufficient for the small molecule-based Click-iT® EdU detection reagent to gain access to the DNA. As a consequence, the Click-iT® EdU imaging kit is not only easy to use, but more accurate and compatible with cell cycle analysis and other intracellular or extracellular targets for truly content-rich results.

This kit is optimized for fluorescence microscopy applications; visit the Click-iT® technology area of our website for kits designed for high-content imaging, fluorescence microplate, or flow cytometry platforms.

Learn more about Click-iT technology >
Find more tools for image-based detection of proliferating cells >

Notes:
The Click-iT® assay can be used on cells in culture or in vivo following EdU administration by feeding or injection methods.
The Click-iT® assay can be used with BrdU in dual pulse experiments by using the anti-BrdU (clone MoBu-1) antibody, which does not cross react with EdU.
The Click-iT® technology is compatible with immunohistochemical, immunocytochemical, and fluorescent dyes that are fixation tolerant or designed for fixed cell labeling.

Click-iT™ TUNEL Colorimetric IHC Detection Kit (Invitrogen™)

The Click-iT™ TUNEL Colorimetric IHC Detection Kit is used to identify apoptotic cells in tissue and cultured cell samples through the use of a small, highly specific labeling moiety and streptavidin-peroxidase conjugation. After incorporation of the labeling moiety into sites of DNA fragmentation, detection is achieved through a catalyzed "click" reaction that adds a biotin group at these sites. The subsequent addition of a streptavidin-peroxidase and peroxidase substrate results in a dark brown signal that can be detected with light microscopy and stored for future analysis.

Find more tools for image-based detection of apoptotic cells >

• Optimized for the detection of apoptotic cells in either tissue or cell samples
• Improved colorimetric TUNEL assay—better label incorporation due to small reactive moiety
• Increased sensitivity—specific label incorporation results in lower background and brighter signal
• Content-rich results—cell morphology, cellular environment, and apoptotic signal are clearly visible
• Flexibility—the assay can be configured for 50 independent TUNEL apoptosis tests

The later stages of apoptosis are characterized by changes in nuclear morphology, chromatin condensation, nuclear envelope degradation, and DNA fragmentation. Terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assays are based on the incorporation of modified dUTPs by terminal deoxynucleotidyl transferase (TdT) at the 3’-OH ends of fragmented DNA. Colorimetric TUNEL assays utilize dUTPs conjugated with a biotin moiety, followed by the addition of a streptavidin-peroxidase conjugate and peroxidase substrate, resulting in a dark brown apoptotic signal. However, colorimetric TUNEL assays are prone to high background, which reduces the sensitivity and specificity of the signal.

The Click-iT TUNEL Colorimetric IHC Detection Kit was developed to address these issues by utilizing a dUTP modified with an alkyne group (a small bio-orthogonal functional group) that enables the nucleotide to be more readily incorporated by TdT. After incorporation, a highly specific click reaction covalently links biotin azide and the alkyne group. Streptavidin-peroxidase (horseradish peroxidase) followed by peroxidase substrate (DAB) are added, allowing colorimetric detection of apoptotic cells. The high degree of labeling specificity inherent in the click technology results in low background and improved detection of apoptotic cells.

The Click-iT TUNEL Colorimetric IHC Detection Kit has been optimized and contains all of the reagents needed for detection of apoptotic cells from either tissue or cell samples. The reagents supplied in this kit can be used to test 50 samples and configured to test from one to 50 samples at a time.

eBioscience™ Mouse Regulatory T Cell Staining Kit #1 (Invitrogen™)

The FJK-16s antibody reacts with mouse/rat Foxp3 also known as FORKHEAD BOX P3, SCURFIN, and JM2; cross reactivity of this antibody to other proteins has not been determined. Foxp3 a 49-55 kDa protein, is a member of the forkhead/winged-helix family of transcriptional regulators, and was identified as the gene defective in ‘scurfy’ (sf) mice. Constitutive high expression of Foxp3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg cells), and ectopic expression of Foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells.

Immunoblotting with FJK-16s antibody has mapped the epitope to amino acids 75-125 of the mouse Foxp3 protein. In the human, this region has been shown to be alternatively spliced at the mRNA level. Both the alternatively-spliced and non-spliced isoforms are present in the CD4+CD25+ subset of lymphocytes. Preliminary RT-PCR experiments have not revealed this alternatively-spliced isoform in mouse splenocytes, suggesting different gene regulation in the mouse and human.

Intracellular staining of mouse splenocytes with FJK-16s using the mouse Foxp3 staining sets and protocol reveals approximately 2% of total splenocytes in the C57Bl/6 strain and approximately 3-5% in the BALB/c mouse strain. Multicolor flow cytometric analysis demonstrates approximately 90% of the CD4+CD25+ cells and 4% of the CD4+CD25- cells staining with FJK-16s. B220+, CD11b+, CD11c+, and Ly6G/Gr-1+ cells do not show significant co-staining with FJK-16s. These data are consistent with a recent report which follows expression of Foxp3 using a GFP knock-in (Fontenot et al, 2005).

FJK-16s cross-reacts with rat Foxp3 This has been demonstrated by intracellular staining of Foxp3 and flow cytometry of rat splenocytes using the same method and reagents as used for mouse tissue. Please note that the CD4 and CD25 antibodies included in this kit only recognize the mouse antigens. For staining rat tissue, please use (CD4 FITC cat. 11-0040 and CD25 APC cat. 17-0390)

The anti-mouse Foxp3 Staining Set has been formulated and optimized for the staining of mouse splenocytes with the FJK-16s monoclonal antibody

Not included:
Isotype controls for anti-CD4 (rat IgG2a, cat. 11-4321) and anti-CD25 (rat IgG1, cat. 17-4301)

Host
Rat

Isotype
IgG2a

Reactivity/Species
Mouse

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

Escherichia coli (K-12 strain) BioParticles™, Texas Red™ conjugate (Invitrogen™)

The Molecular Probes® BioParticles® product line consists of a series of fluorescently labeled, heat- or chemically killed bacteria and yeast in a variety of sizes, shapes, and natural antigenicities. These fluorescent BioParticles® products have been employed to study phagocytosis by fluorescence microscopy, quantitative spectrofluorometry, and flow cytometry.

We offer E. coli (K-12 strain), S. aureus (Wood strain, without protein A) and zymosan (S. cerevisiae) BioParticles® conjugates covalently labeled with a variety of different fluorophores (special care has been taken to remove free dye after conjugation). Unlike the fluorescence of fluorescein-labeled BioParticles® conjugates, which is partially quenched in acidic environments, the fluorescence of the Alexa Fluor®, BODIPY® FL, tetramethylrhodamine and Texas Red® dye conjugates is uniformly intense over the pH range from 3 to 10.

BioParticles Specifications:
• Label (Ex/Em): Texas Red® (~595/615 nm)
• Particle: E. coli (K-12 strain)
Opsonizing reagent available


Using BioParticles Products
BioParticles® conjugates are provided as lyophilized powders. There are approximately 3 x 108 E. coli or S. aureus particles per mg solid and approximately 2 x 107 zymosan particles per mg solid. BioParticles® conjugates can be reconstituted in the buffer of your choice for use in phagocytosis assays. The fluorescence of BioParticles® conjugates that are bound to the surface of the cell (but not internalized) can be quenched by ethidium bromide, trypan blue, or other quenchers. In addition to cellular applications, fluorescent BioParticles® conjugates may be effective as flow cytometry calibration references when sorting bacteria and yeast mutants. These small particles may also be useful references for light scattering studies because their sizes and shapes differ in characteristic ways.

Find More BioParticles® Products
We offer a large range of dye-labeled and unlabeled E. coli (K-12 strain), S. aureus (Wood strain, without protein A), and zymosan (S. cerevisiae) BioParticles® products. Find out about these products and their applications by reviewing Probes for Following Receptor Binding and Phagocytosis—Section 16.1 in the Molecular Probes® Handbook.

For pH-sensitive endocytosis assays, see our pHrodo™ BioParticles® conjugates.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

Arcturus™ Paradise™ PLUS qRT-PCR Kit, no solvents (Applied Biosystems™)

The ARCTURUS® Paradise® PLUS Reagent System enables unprecedented gene expression analysis of millions of previously inaccessible samples. The Paradise® PLUS Reagent System is an integrated solution for gene expression studies from formalin-fixed, paraffin-embedded (FFPE) tissues, including reagents for tissue staining, RNA isolation and RNA amplification.

Key product features:
• Accelerated discovery process – unlock the tissue archives to access previously inaccessible samples
• Streamlined application – integrate seamlessly into current workflow
• Efficient workflow – secure downstream success with the Paradise® QC Kit
• Comprehensive system – benefit from the only complete system for formalin-fixed samples

Unlock the Tissue Archives
Currently, tens of millions of tissue samples with known outcomes are available in tissue archives. The Paradise® PLUS Reagent system enables microarray and qRT-PCR analysis of these previously inaccessible samples, allowing retrospective studies to accelerate discoveries. Achieve unprecedented results in just six steps (Figure 1).

Integrate Seamlessly into Current Workflow
The Paradise® PLUS Reagent System is compatible with all major gene expression platforms. Robust and reproducible results are achieved using tools and techniques commonly employed in today’s research laboratories.

Secure Downstream Success with the Paradise® QC Kit
Not all FFPE samples contain high quality RNA. Identify the best samples for your studies using a simple, real-time PCR research technique, and then proceed with confidence using these pre-qualified samples.

Benefit from the Only Complete System for Formalin-fixed Samples
The Paradise® PLUS Reagent System is the only available solution that provides all the components necessary to prepare RNA from formalin-fixed, paraffin-embedded tissue samples for gene expression analysis. To ensure successful gene expression results, the system includes reagents optimized for exceptional recovery of RNA and superior reverse transcription and RNA amplification (Figure 3).

Integrated Systems for Microgenomics
The Paradise® PLUS Reagent System is validated as part of ARCTURUS®' complete Systems for Microgenomics®, an integrated solution for utilizing small quantities of RNA for gene expression analysis, and features the ARCTURUSXT Laser Capture Microdissection (LCM) Instrument to procure pure cell populations. ARCTURUS®' Systems for Microgenomics enable accurate and sensitive microarray assays that reveal molecules otherwise obscured in whole tissue samples or cell mixtures.

For Research Use Only. Not for use in diagnostics procedures.

pHrodo™ Green Zymosan Bioparticles™ Conjugate for Phagocytosis (Invitrogen™)

New pH-sensitive pHrodo® Green dye conjugates, like our pHrodo® Red dye conjugates, give faster and more accurate results than any other phagocytosis assay. pHrodo® Green conjugates are non-fluorescent outside the cell at neutral pH, but fluoresce brightly green at acidic pH such as in phagosomes. Get faster staining and more accurate results—without the need for wash steps or quencher dye.

• Specific detection of phagocytosis and endocytosis
• Reduced signal variability and improved timing in sensitive experiments
• Enough to do 100 tests when using 100 µL in each well of a 96-well plate
• Multiplex with other compatible dyes, such as red florescent protein, TMRM, NucBlue™ Hoechst, and CellROX® Deep Red

The fluorescence of the novel pHrodo® Green dye dramatically increases as pH decreases from neutral to acidic, making it an ideal tool to study phagocytosis and its regulation by drugs and/or environmental factors. The lack of fluorescence outside the cell eliminates the need for wash steps and quencher dyes.

Use the ready-made pHrodo® Green Zymosan BioParticles® Conjugate in imaging, high content screening, high throughput screening, and flow applications. pHrodo® Green dye is also available as a conjugate of E. coli BioParticles®, S. aureus BioParticles®, or dextran 10,000. To create other conjugates, such as antibody conjugates, use pHrodo® Green STP ester or pHrodo® Green maleimide.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

Amplex™ Red Glutamic Acid/Glutamate Oxidase Assay Kit (Invitrogen™)

The Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit provides a sensitive and simple method for detecting glutamic acid/glutamate oxidase activity using a fluorescence microplate reader or fluorometer.

See our complete line of Fluorescence Microplate assays.

• Detect concentrations as low as 10 nM L-glutamic acid or 40 µU/mL of purified L-glutamate oxidase
• Format allows for multiple time point measurements
• Designed for minimal autofluorescence interference

The Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit provides an ultrasensitive method for detecting glutamic acid or for continuously monitoring glutamate oxidase activity using a fluorescence microplate reader or fluorometer. In the assay, L-glutamic acid is oxidized by glutamate oxidase to produce α-ketoglutarate, NH3, and hydrogen peroxide. L-alanine and L-glutamate–pyruvate transaminase are included in the reaction to regenerate L-glutamic acid by transamination of α-ketoglutarate, resulting in multiple cycles of the initial reaction and a significant amplification of the hydrogen peroxide produced. The hydrogen peroxide reacts with 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red reagent) in a 1:1 stoichiometric ratio in a reaction catalyzed by horseradish peroxidase (HRP) to generate the highly fluorescent product resorufin.

Because resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, there is little interference from autofluorescence in most biological samples.

Use Amplex® Red Assays for a Broad Range of Investigations
A wide variety of validated Amplex® Red assays are available for studying cell signaling and lipids, neurobiology, inflammation and immune function, and metabolism. We also offer Amplex® UltraRed Reagent (Cat. No. A36006), a second-generation reagent providing greater sensitivity and brighter fluorescence, and the Amplex® Red/UltraRed Stop Reagent (Cat. No. A33855). The Amplex® Red/UltraRed Stop Reagent provides convenience and control by allowing the fluorescence signal-generating reaction to be terminated at a user-determined time point. After addition of the stop reagent, the fluorescence signal remains stable for at least three hours. Custom assay design and packaging are also available.

CellTrace™ Violet Cell Proliferation Kit, for flow cytometry (Invitrogen™)

CellTrace™ Violet Cell Proliferation Kit is used for in vitro and in vivo labeling of cells to trace multiple generations using dye dilution by flow cytometry.

• Superior performance—bright, single-peak staining enables visualization of multiple generations
• Long-term signal stability—well-retained in cells for several days post stain
• Versatile—multiple colors available to easily combine with antibodies or markers of cell function, such as GFP
• Simple, robust staining protocol

View a selection guide for all CellTrace™ Cell Proliferation Kits for flow cytometry.

Superior fluorescent staining
Successful proliferation analysis by dye dilution (see figure below) requires an extremely bright dye to distinguish fluorescently labeled cells from auto-fluorescence after several cell divisions. The intense fluorescent staining provided by CellTrace™ Violet dye enables the visualization of ten or more generations of proliferating cells before the signal is overwhelmed by intrinsic cellular auto-fluorescence. Consistent, homogeneous staining results in very little fluorescence variation between cells in a population, so distinct generations can be seen without any requirement for complex modeling software.

Long-term signal retention
Unlike stains that label the lipid membrane of cells, CellTrace™ Violet stain easily crosses the plasma membrane and covalently binds inside cells where the stable, well-retained fluorescent dye provides a consistent signal, even after several days in a cell culture environment. CellTrace™ Violet dye binds covalently to all free amines on the surface and inside of cells and shows little cytotoxicity, with minimal observed effect on the proliferative ability or biology of cells.

Easy multiplexing with other fluorophores
The violet excitation and narrow emission of CellTrace™ Violet dye make it ideal for multiplexing due to the limited spectral overlap with other common dyes (Alexa Fluor™ 488, FITC, and RPE) and fluorescent proteins (Green Fluorescent Protein (GFP) and mCherry) (see Fluorescence Spectra for CellTrace™ Violet stain below).

Simple, robust staining protocol
The CellTrace™ Violet Cell Proliferation Kit contains convenient single-use vials of dry dye to permit small-scale experiments without preparing excess quantities of dye. A stock solution is prepared by dissolving the contents of a vial in anhydrous DMSO prior to use. To stain 1 mL of cells in protein-free medium, 1 µL of this stock solution is typically used. Cells should be stained for 20 minutes at room temperature with gentle agitation. A brief wash with complete medium will then quench any dye remaining in solution.

eBioscience™ Annexin V-FITC Apoptosis Detection Kit (Invitrogen™)

Annexins are a family of calcium-dependent phospholipid-binding proteins, which bind to phosphatidylserine (PS) to identify apoptotic cells. In healthy cells, PS is predominantly located along the cytosolic side of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution in the phospholipid bilayer and translocates to the extracellular membrane, which is detectable with fluorescently labeled Annexin V. In early stages of apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI) and 7-AAD, therefore cells which display only Annexin V staining (PI/7-AAD negative) are in early stages of apoptosis. During late-stage apoptosis, loss of cell membrane integrity allows Annexin V binding to cytosolic PS, as well as cell uptake of PI and 7-AAD. Annexin V staining, paired with 7-AAD or PI is widely used to identify apoptotic stages by flow cytometry.

Host
E.coli

Conjugate
FITC

Laser
Blue Laser

Emit
520 nm

Excite
488 nm

Purity
> 98% pure

Molecular Mass
35.8 kDa

Reported Application
Flow Cytometric Analysis

eBioscience™ BrdU Kit for IHC/ICC Immunofluorescence eFluor™ 660 (Invitrogen™)

This BrdU Kit for immunohistochemistry (IHC)/immunocytochemistry (ICC) with immunofluorescence detection contains the necessary reagents and buffers for identifying and examining proliferating cells by immunohistochemical or immunocytochemical analysis. Cells are labeled in vitro or in vivo with 5-bromo-2’-deoxyuridine (BrdU), a synthetic analog of thymidine, which is incorporated into DNA in place of thymidine during the S-phase of the cell cycle. Following fixation and antigen retrieval steps, cells or tissue sections are stained for BrdU incorporation and visualized using a streptavidin-conjugated fluorophore. This kit has been optimized for IHC with both frozen and formalin-fixed paraffin embedded BrdU-labeled mouse intestine and ICC of BrdU-pulsed HeLa cells grown on culture slides.

Conjugate
eFluor 660

Laser
Red Laser

Emit
668 nm

Excite
633 - 647 nm

Reported Application
Immunohistochemical Staining of Formalin-Fixed Paraffin Embedded Tissue Sections, Immunohistochemical Staining of Frozen Tissue Sections, Immunocytochemistry, Microscopy

XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) (Invitrogen™)

XTT is used to assess cell viability as a function of redox potential. Actively respiring cells convert the water-soluble XTT to a water-soluble, orange colored formazan product. Unlike MTT, XTT does not require solubilization prior to quantitation, thereby reducing the assay time in many viability assay protocols. Moreover, the sensitivity of the XTT reduction assay is reported to be similar to or better than that of the MTT reduction assay.

BacLight™ Green Bacterial Stain (Invitrogen™)

The BacLight Green (Cat. no. B-35000) and BacLight Red (Cat. no. B-35001) bacterial stains are fluorescent labeling reagents for detecting and monitoring bacteria. These two dyes are not nucleic acid stains. Bacteria stained with the BacLight Green and BacLight Red bacterial stains exhibit bright green and red fluorescence (absorption/emission ~480/516 and ~581/644 nm, respectively), and can be resolved using the appropriate flow cytometric channels or fluorescence microscopy filters. The BacLight bacterial stains are compatible with formaldehyde or alcohol fixation methods.

View additional information about all microbiological analysis products.

Arcturus™ Paradise™ PLUS 2 Round Kit, amino allyl, no solvents (Applied Biosystems™)

The ARCTURUS® Paradise® PLUS Reagent System permits unprecedented gene expression analysis of millions of previously inaccessible samples. It is an integrated solution for gene expression studies from formalin-fixed, paraffin-embedded (FFPE) tissues, including reagents for tissue staining, RNA isolation and RNA amplification. Amplify RNA from as little as 5 ng of fixed RNA using methods that have been specially optimized to overcome the challenges of cross-linked templates.

This kit provides reagents for 2-round in-vitro transcription, and aminoallyl aRNA labeling.

Key product features:
• Accelerated discovery process – unlock the tissue archives to access previously inaccessible samples
• Simplified procedure – achieve unprecedented results in six steps
• Streamlined application – integrate seamlessly into current workflow
• Efficient workflow – secure downstream success with the Paradise® QC Kit
• Comprehensive system – benefit from the only complete system for formalin-fixed samples

Unlock the Tissue Archives
Currently, tens of millions of tissue samples with known outcomes are available in tissue archives. The Paradise® PLUS Reagent System enables microarray and qRT-PCR analysis of these previously inaccessible samples, allowing retrospective studies to accelerate discoveries.

Achieve Unprecedented Results in Six Steps
Only six steps are required to take you from sample to results (See Figure 1).

Integrate Seamlessly into Current Workflow
The Paradise® PLUS Reagent System is compatible with all major microarray platforms. Robust and reproducible results are achieved using tools and techniques commonly employed in today’s research laboratories (See Figures 2, 3, 4, and 5).

Secure Downstream Success with the Paradise® QC Kit
Not all FFPE samples contain high quality RNA. Identify the best samples for your studies using a simple, real-time PCR research technique, and then proceed with confidence using these pre-qualified samples (See Figure 6).

Benefit from the Only Complete System for Formalin-fixed Samples
The Paradise® PLUS Reagent System is the only available solution that provides all the components necessary to prepare RNA from formalin-fixed, paraffin-embedded tissue samples for gene expression analysis. To ensure successful microarray results, the system includes reagents optimized for exceptional recovery of RNA and superior RNA amplification.

Integrated Systems for Microgenomics
The Paradise PLUS Reagent System is a validated as part of the Arcturus® Complete System for Microgenomics®, an integrated solution for utilizing small quantities of RNA for gene expression analysis. This system features the ARCTURUSXT™ Laser Capture Microdissection (LCM) Instrument to procure pure cell populations. Arcturus Systems for Microgenomics enable accurate and sensitive microarray assays that reveal molecular signatures otherwise obscured in whole tissue samples or cell mixtures.

For Research Use Only. Not for use in diagnostics procedures.

Amplex™ Red Galactose/Galactose Oxidase Assay Kit (Invitrogen™)

The Amplex® Red Galactose/Galactose Oxidase Assay Kit provides a sensitive and simple method for detecting galactose oxidase activity or galactose using a fluorescence microplate reader or fluorometer.

See our complete line of Fluorescence Microplate assays.

• Detect concentrations as low as 2 mU/mL of galactose oxidase activity or 4 µM galactose
• Format allows for multiple time point measurements
• Designed for minimal autofluorescence interference

Galactose oxidase is an important tool in the detection of galactose in biological fluids. The Amplex® Red Galactose/Galactose Oxidase Assay Kit provides an ultrasensitive method for detecting galactose or for monitoring galactose oxidase activity. Galactose oxidase catalyzes the oxidation of galactose at the C6 position and generates hydrogen peroxide. In the presence of horseradish peroxidase (HRP), hydrogen peroxide reacts in a 1:1 stoichiometric ratio with the Amplex® Red reagent to generate the red-fluorescent compound resorufin.

Because resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, there is little interference from autofluorescence in most biological samples.

Use Amplex® Red Assays for a Broad Range of Investigations
A wide variety of validated Amplex® Red assays are available for studying cell signaling and lipids, neurobiology, inflammation and immune function, and metabolism. We also offer Amplex® UltraRed Reagent (Cat. No. A36006), a second-generation reagent providing greater sensitivity and brighter fluorescence, and the Amplex® Red/UltraRed Stop Reagent (Cat. No. A33855). The Amplex® Red/UltraRed Stop Reagent provides convenience and control by allowing the fluorescence signal-generating reaction to be terminated at a user-determined time point. After addition of the stop reagent, the fluorescence signal remains stable for at least three hours. Custom assay design and packaging are also available.

eBioscience™ Essential Human Treg Phenotyping Kit (Invitrogen™)

The Invitrogen eBioscience Essential Human Treg Phenotyping Kit is designed for flow cytometry-based identity testing of regulatory T-cells (Tregs). It leverages high-quality eBioscience antibodies, isotype controls, and fixation/permeabilization reagents (for the intracellular marker FOXP3) and includes a ready-to-use protocol for experimental setup, gating strategy, and analysis.

Benefits include:
Proven performance―antibodies and supporting reagents designed for flow cytometry
Highly referenced antibodies―includes one of the most referenced clones for FOXP3
Turnkey kit and protocol―contains reagents and standardized gating recommendations needed to perform flow cytometry characterization

eBioscience™ Annexin V Apoptosis Detection Kit PerCP-eFluor™ 710 (Invitrogen™)

Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS) in all mammalian species. Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.

In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD, or Fixable Viability Dyes such as eFluor™ 660 or eFluor™ 780. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).

Due to the emission spectrum of PerCP-eFluor™ 710, the Annexin V Apoptosis Detection Kit PerCP-eFluor™ 710 is not compatible with propidium iodide and 7-AAD. It is recommended to substitute a Fixable Viability Dye such as eFluor™ 660 or eFluor™ 780 in their place.

Not included:
Fixable Viability Dye eFluor™ 660 (cat. 65-0864)
Fixable Viability Dye eFluor™ 780 (cat. 65-0865)

Note: Fixable Viability Dye eFluor™ 450 is not recommended for use with Annexin V Apoptosis Detection Kits.

Reported Application
Flow Cytometric Analysis

Vybrant™ Apoptosis Assay Kit #4, YO-PRO™-1/Propidium Iodide (Invitrogen™)

The Vybrant Apoptosis Assay Kit #4 detects apoptosis on the basis of changes that occur in the permeability of cell membranes. This kit contains ready-to-use solutions of both YO-PRO-1 and propidium iodide nucleic acid stains. YO-PRO-1 stain selectively passes through the plasma membranes of apoptotic cells and labels them with moderate green fluorescence. Necrotic cells are stained red-fluorescent with propidium iodide.

View a selection guide for all apoptosis assays for flow cytometry.

LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit, for 405 nm excitation (Invitrogen™)

The LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit is used to determine the viability of cells prior to the fixation and permeabilization required for intracellular antibody staining or prior to elimination of biohazardous materials using formaldehyde fixation. This kit has been optimized and validated for use with a violet laser flow cytometer.

• Stable—dyes are freeze dried in separate vials to maintain stability
• Robust—staining pattern is the same before and after fixation
• Low compensation—minimal spectral overlap between other fluorophores

View a selection guide for all fixable viability dyes for flow cytometry.

Stable
Unlike products that are sold in solution, the LIVE/DEAD™ Fixable Violet Stain has been conveniently packaged in 40-test vials to help ensure the stability and performance of the dye over time. Amine reactive dyes in solution will lose their effectiveness over a short period of time, therefore it is recommended to completely use the vial once rehydrated. If this is not possible, aliquot the vials in small volumes and store at -80°C, avoiding freeze-thaw cycles.

Robust
Dead cell discriminator stains can lose sensitivity after treatment with fixatives such as formaldehyde or ethanol-based fixation methods required for intracellular phosphorylation studies. The LIVE/DEAD™ Fixable Violet Stain is an amine reactive dye that binds covalently to intracellular and extracellular amines, and the staining pattern is preserved following formaldehyde fixation.

Low compensation
The LIVE/DEAD™ Fixable Violet Stain was selected based on its fluorescent properties to minimize compensation between other violet dyes and dyes that excite off of the 488 nm blue laser. The violet-fluorescent reactive dye has an excitation maximum of ~416 nm and is excited well with the 405 nm violet laser, and it has an emission maxima of ~451 nm, so it can be collected in the first channel on most violet laser flow cytometers.

How it works
In cells with compromised membranes, the dye reacts with free amines both in the cell interior and on the cell surface, yielding intense fluorescent staining. In viable cells, the dye's reactivity is restricted to the cell-surface amines, resulting in less intense fluorescence. The difference in intensity is typically greater than 50-fold between live and dead cells, allowing for easy discrimination.

Colors available
LIVE/DEAD™ Fixable Dead Cell Stains are available in a wide variety of colors to meet your multi-color panel needs.

Click-iT™ EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit (Invitrogen™)

The Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit provides a simplified, more robust assay for analyzing DNA replication in proliferating cells as compared to traditional BrdU methods. Newly synthesized DNA is analyzed using the 633/5 nm laser of the flow cytometer.

Accurate--Superior Results compared to BrdU Assays
Fast--Results in as little as 90 minutes
Economical--More assays per kit

View selection guide for all Click-iT™ EdU and Click-iT™ Plus EdU assays for flow cytometry.

An Advanced Method Giving You Results Superior to BrdU
The most accurate method of proliferation analysis is direct measurement of DNA synthesis. Originally, this was performed through incorporation of radioactive nucleosides, i.e., 3H-thymidine. This method was replaced by antibody-based detection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT® EdU Flow Cytometry Assay Kits are novel alternatives to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog which is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry: a copper catalyzed covalent reaction between an azide and an alkyne. In this application, the alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to Alexa Fluor ® 488, Alexa Fluor® 647, or Pacific BlueTM dyes. Standard flow cytometry methods are used for determining the percentage of S-phase cells in the population (Fig 1).

Mild Conditions Allow Use with Cell Cycle Dyes and Antibodies
The advantages of Click-iT® EdU labeling are readily evident while performing the assay (Fig 2). The small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions, while standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT® detection reagent to gain access to the DNA. This is in contrast to BrdU assays that require DNA denaturation (using HCl, heat, or digestion with DNase) to expose the BrdU so that it may be detected with an anti-BrdU antibody. Sample processing for the BrdU assay can result in signal alteration of the cell cycle distribution as well as destruction of antigen recognition sites when using the HCl method. In contrast, the easy-to-use EdU cell proliferation kit is compatible with cell cycle dyes. This EdU assay kit can also be multiplexed with antibodies against surface and intracellular markers.

Quick and Simple Protocol
The Click-iT® EdU protocol is based on the aldehyde fixation and detergent permeabilization steps for immunohistochemical antibody labeling, but EdU is compatible with other
fixation/permeabilization agents including saponin and methanol. In just five steps you’ll be ready to analyze your cell proliferation data:

• Treat cells with EdUM
• Fix and permeabilize cells
• Detect S-phase cells with Click-iT® detection cocktail for 30 min
• Wash once
• Analyze

Get Accurate Results Economically
By increasing the number of assays per kit, the Click-IT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit is less expensive than the traditional BrdU assays making them ideal for large experiments.

For research use only. Not intended for any human or animal therapeutic of diagnostic use.

Histogene™ Staining Solution (Applied Biosystems™)

This staining solution is for use with the ARCTURURS® HistoGene® LCM Frozen Section Staining Kit.

Designed as a fast-penetrating stain, it provides excellent contrast while preserving nucleic acid integrity and retaining low-abundance mRNAs in frozen tissue sections intended for laser capture microdissection (LCM).

For Research Use Only. Not for use in diagnostics procedures.

Escherichia coli (K-12 strain) BioParticles™, Alexa Fluor™ 488 conjugate (Invitrogen™)

The Molecular Probes® BioParticles® product line consists of a series of fluorescently labeled, heat- or chemically killed bacteria and yeast in a variety of sizes, shapes, and natural antigenicities. These fluorescent BioParticles® products have been employed to study phagocytosis by fluorescence microscopy, quantitative spectrofluorometry, and flow cytometry.

We offer E. coli (K-12 strain), S. aureus (Wood strain, without protein A) and zymosan (S. cerevisiae) BioParticles® conjugates covalently labeled with a variety of different fluorophores (special care has been taken to remove free dye after conjugation). Unlike the fluorescence of fluorescein-labeled BioParticles® conjugates, which is partially quenched in acidic environments, the fluorescence of the Alexa Fluor®, BODIPY® FL, tetramethylrhodamine and Texas Red® dye conjugates is uniformly intense over the pH range from 3 to 10.

BioParticles Specifications:
• Label (Ex/Em): Alexa Fluor® 488 (~495/519 nm)
• Particle: E. coli (K-12 strain)
Opsonizing reagent available


Using BioParticles Products
BioParticles® conjugates are provided as lyophilized powders. There are approximately 3 x 108 E. coli or S. aureus particles per mg solid and approximately 2 x 107 zymosan particles per mg solid. BioParticles® conjugates can be reconstituted in the buffer of your choice for use in phagocytosis assays. The fluorescence of BioParticles® conjugates that are bound to the surface of the cell (but not internalized) can be quenched by ethidium bromide, trypan blue, or other quenchers. In addition to cellular applications, fluorescent BioParticles® conjugates may be effective as flow cytometry calibration references when sorting bacteria and yeast mutants. These small particles may also be useful references for light scattering studies because their sizes and shapes differ in characteristic ways.

Find More BioParticles® Products
We offer a large range of dye-labeled and unlabeled E. coli (K-12 strain), S. aureus (Wood strain, without protein A), and zymosan (S. cerevisiae) BioParticles® products. Find out about these products and their applications by reviewing Probes for Following Receptor Binding and Phagocytosis—Section 16.1 in the Molecular Probes® Handbook.

For pH-sensitive endocytosis assays, see our pHrodo™ BioParticles® conjugates.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

Amplex™ Red Xanthine/Xanthine Oxidase Assay Kit (Invitrogen™)

The Amplex Red Xanthine/Xanthine Oxidase Assay Kit provides a sensitive and simple fluorometric method for detecting as little as 200 nM xanthine or hypoxanthine or as little as 0.1 mU/mL of xanthine oxidase activity in a purified system with a 100 µL assay volume.

eBioscience™ Mouse Hematopoietic Lineage Biotin Panel (Invitrogen™)

The eBioscience Mouse Hematopoietic Lineage Flow Panel contains 5 biotinylated antibodies that can be used to identify, enrich and/or deplete cells committed to the T, B, NK, myeloid and erythroid lineages based on their cell surface antigen expression.

Reactivity/Species
Mouse

Reported Application
Flow Cytometric Analysis

Click-iT™ EdU Alexa Fluor™ 594 HCS Assay (Invitrogen™)

The Click-iT® EdU Alexa Fluor® 594 HCS Assay is a superior alternative to traditional proliferation assays that is optimized for high-content imaging applications. In this assay the modified thymidine analogue EdU is efficiently incorporated into newly synthesized DNA and fluorescently labeled with a bright, photostable Alexa Fluor® dye in a fast, highly-specific click reaction. This fluorescent labeling of proliferating cells is accurate and compatible with antibody methods due to the mild click protocol.

• Simple—works the first time, every time, in less time
• Efficient—no denaturation steps or harsh treatment required
• Content-rich results—better preservation of cell morphology, antigen structure, and DNA integrity
• Consistent—not dependent on variable antibody lots for detection

The most accurate proliferation-detection methods are based on the incorporation and measurement of nucleoside analogues in newly synthesized DNA, with bromodeoxyuridine (BrdU) a commonly used analogue. BrdU-labeled DNA is quantitated using anti-BrdU antibodies following DNA denaturation by harsh methods (HCl, heat, or enzymes) to expose the BrdU molecules. This step is time consuming and difficult to perform consistently. The harsh treatment can also adversely effect sample integrity and quality, which makes co-staining with other antibodies challenging.

Superior Proliferation Methodology
The Click-iT® EdU Alexa Fluor® 594 HCS Assay provides a superior alternative to BrdU assays for measuring cell proliferation. EdU (5-ethynyl-2'-deoxyuridine) is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis. With Click-iT® EdU, mild fixation and detergent permeabilization is sufficient for the small molecule-based Click-iT® EdU detection reagent to gain access to the DNA. As a consequence, the Click-iT® EdU HCS assay is not only easy to use, but more accurate and compatible with cell cycle analysis and other intracellular or extracellular targets for truly content-rich results.

The kit is optimized for high-content imaging applications; visit the Click-iT® technology area of our website for kits designed for fluorescence microscopy, microplate, or flow cytometry platforms.

Learn more about Click-iT® technology

Notes:
The Click-iT® assay can be used on cells in culture or in vivo following administration of EdU by feeding or injection methods.
The Click-iT® assay can be used with BrdU in dual pulse experiments by using the ant-BrdU (clone MoBu-1) antibody, which does not cross react with EdU.
The Click-iT® technology is compatible with immunohistochemical, immunocytochemical, and fluorescent dyes that are fixation tolerant or designed for fixed-cell labeling.

Pierce™ Peroxidase IHC Detection Kit (Thermo Scientific™)

The Thermo Scientific Pierce Peroxidase IHC Detection Kit bundles the Pierce Metal Enhanced DAB Substrate Kit with all necessary components to stain, counterstain and preserve experimental results with frozen or paraffin tissue sections.

Features of the Peroxidase IHC Detection Kit:

Incredibly sensitive—fifty times more sensitive than the traditional DAB (3,3'-diaminobenzidine) method and 30 times more sensitive than other metal-intensified versions of DAB
Low background, high intensity, large dynamic range—gives a crisp dark brown-black precipitate that's more intense than the dull brown precipitate observed when using DAB without enhancement; substrate delivers increased intensity with almost nonexistent background
Easy-to-use, two-component system—mix the two liquid components and the working solution is ready-to-use; sufficient substrate and other staining reagents to treat up to 500 slides
Six-hour working solution stability—unlike other DAB substrates that must be used immediately, the working solution is stable for more than six hours at room temperature
Complete kit—saves time in reagent ordering, gathering and validating reagents for the IHC application; saves money compared to purchasing of all components separately

This immunohistochemical staining kit bundles the Pierce Metal Enhanced DAB Substrate Kit with all necessary components to stain, counter-stain and preserve experimental results with frozen or paraffin tissue section that have been probed with peroxidase enzyme conjugates. The Pierce Peroxidase IHC Detection Kit eliminates the need to procure numerous reagents from various sources, resulting in a time- and cost-effective way to perform peroxidase-based tissue staining. Harris Hematoxylin nuclear stain and mounting medium allow counter-staining and tissue preservation, respectively. The detection method is compatible with direct or avidin-biotin-complex (ABC) probing strategies.

Applications:
• Tissue immunohistochemistry procedures designed for DAB precipitating substrate
• DAB staining of fixed paraffin sections, smears and frozen section preparations
• Immunohisto- or immunocyto-staining using the metal-enhanced DAB substrate

Click-iT™ Plus EdU Alexa Fluor™ 594 Flow Cytometry Assay Kit (Invitrogen™)

The Click-iT Plus EdU Alexa Fluor 594 Flow Cytometry Assay Kit provides a simplified, more robust assay for analyzing DNA replication in proliferating cells compared to traditional BrdU methods. Newly synthesized DNA is analyzed using the 532- or 561-nm laser of the flow cytometer. The Click-iT Plus formulation is compatible with standard fluorophores, including R-PE and R-PE tandems, as well as fluorescent proteins.

• Multiplexable—compatible with R-PE (and tandems) and fluorescent proteins
• Accurate—superior results compared to BrdU assays
• Fast—results in as little as 60 minutes

View selection guide for all Click-iT EdU and Click-iT Plus EdU assays for flow cytometry.

Multiplexable
Click-iT Plus Alexa Fluor 594 EdU assays can be used in conjunction with R-PE and R-PE tandems, as well as fluorescent proteins such as GFP and mCherry. The Alexa Fluor 594 labeling reagent is excited readily at either 532 or 561 nm and emits at 615 nm.

An Advanced Method Giving You Results Superior to BrdU
The most accurate method of proliferation analysis is direct measurement of DNA synthesis. Originally, this was performed through incorporation of radioactive nucleosides. This method was replaced by antibody-based detection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT Plus EdU Flow Cytometry assay is a novel alternative to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog that is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry, which is a copper-catalyzed covalent reaction between an azide and an alkyne. In this application, the alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to the Alexa Fluor dye. Standard flow cytometry methods are used for determining the percentage of S-phase cells in the population.

Mild Conditions Allow Use with Cell Cycle Dyes and Antibodies
The small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions, while standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT Plus detection reagent to gain access to the DNA. This is in contrast to BrdU assays that require DNA denaturation (using HCl, heat, or digestion with DNase) to expose the BrdU so that it can be detected with an anti-BrdU antibody. Sample processing for the BrdU assay can result in signal alteration of the cell cycle distribution, as well as destruction of antigen recognition sites when using the HCl method. In contrast, the easy-to-use Click-iT Plus EdU assay is compatible with cell cycle dyes. The Click-iT Plus EdU assay can also be multiplexed with antibodies against surface and intracellular markers, as well as conjugates labeled with standard fluorophores including R-PE, R-PE tandems, and fluorescent proteins (GFP and mCherry).

Quick and Simple Protocol
The Click-iT Plus EdU protocol is based on the aldehyde fixation and detergent permeabilization steps for immunohistochemical antibody labeling. However, EdU is compatible with other fixation/permeabilization agents including saponin and methanol. In just five steps you’ll be ready to analyze your cell proliferation data:

1. Treat cells with EdU.
2. Fix and permeabilize cells.
3. Detect S-phase cells with Click-iT Plus detection cocktail for 30 min.
4. Wash once.
5. Analyze.

Results can be seen in as little as 60 minutes in some circumstances, but we recommend 90 minutes for all applications.