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CyQUANT™ NF Cell Proliferation Assay (Invitrogen™)

The CyQUANT® NF Cell Proliferation Assay provides a fast and sensitive method for counting cells in a population and measuring proliferation in microplate format.

Features of the CyQUANT® NF Cell Proliferation Assay:

• More sensitive than MTT or AlamarBlue® assays
• Linear detection range from 100 to 20,000 cells per well (96-well microplate)
• Assays can be completed in one hour

A Rapid and simple method for measuring cell proliferation
The CyQUANT® NF Cell Proliferation Assay does not require cell lysis, long incubations, radioactivity, or removal of stain from cells. The CyQUANT® NF assay eliminates the freeze-thaw cell lysis step of the original CyQUANT® cell proliferation assay by using a cell-permeant DNA-binding dye in combination with a plasma membrane permeabilization reagent. The CyQUANT® NF Cell Proliferation Assay can be used in either 96-well or 384-well microplate formats, and is available in two configurations: a 200 assay kit (C35007) for smaller sample sizes, and a 1000 assay kit (C35006) for high-throughput applications.

AlamarBlue® is a registered trademark of TREK Diagnostic Systems.

CellEvent™ Caspase-3/7 Green Detection Reagent (Invitrogen™)

CellEvent™ Caspase-3/7 Green Detection Reagent is a novel fluorogenic substrate for activated caspase-3/7 that is compatible with both live cell and fixed imaging, with absorption/emission maxima of ~502/530 nm. Activation of caspase-3 is an essential event during apoptosis, making this an optimized reagent for analysis of apoptotic cells.

See more tools for detection of apoptotic cells >

• Optimized caspase-3/7 substrate for apoptosis analysis
• Easy to use—add and read; no cell lysis required
• Use for time course measurements; easily select the time point of interest
• Compatible with both live cell fluorescence-imaging and formaldehyde-based fixation methods
• Multiplex enabled—combine with other fluorescent reagents to confirm apoptosis in the same cell or cell population

Substrate specifics
CellEvent Caspase-3/7 Green Detection Reagent is a four-amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye with absorption/emission maxima of ~502/530 nm. The DEVD peptide sequence is a cleavage site for caspase-3/7, and the conjugated dye is non-fluorescent until cleaved from the peptide and bound to DNA.

Principles of action
CellEvent Caspase-3/7 Green Detection Reagent is intrinsically non-fluorescent as the DEVD peptide inhibits the ability of the dye to bind to DNA. However, after activation of caspase-3/7 in apoptotic cells, the DEVD peptide is cleaved, enabling the dye to bind to DNA and produce a bright, fluorogenic response. The fluorescent emission of the dye when bound to DNA is ~530 nm and can be observed using a standard FITC filter set.

Simple three-step protocol
To use CellEvent Caspase 3/7 Detection Reagent, simply add it to cells, incubate 30 minutes, and visualize (see figure). Apoptotic cells with activated caspase-3/7 will have bright green nuclei, while cells without activated caspase 3/7 will have minimal fluorescent signal (see figure).

Assay versatility allows for live cell detection and fixation
This robust assay enables the examination of caspase-3/7 activation in live cells. Furthermore, since wash steps are not necessary for detection, fragile apoptotic cells typically lost during wash steps are preserved. Importantly, the fluorescent signal from CellEvent Caspase 3/7 Detection Reagent can survive fixation and permeabilization. This allows for the flexibility to perform end-point assays and probe for other proteins of interest using immunocytochemistry.

eZKine™ CD8 Activation 1 Whole Blood Intracellular Cytokine Kit (Invitrogen™)

This eZKine CD8 Activation 1 Whole Blood Intracellular Cytokine Kit is designed to rapidly identify cytokine producing T lymphocytes of the CD8+ T cell lineage after stimulation of whole peripheral blood samples. Stimulation of whole blood in the presence of Protein Transport Inhibitor Cocktail (cat. 00-4980, not included) is followed by red blood cell lysis and fixation in a single step and subsequent permeabilization. Samples are then ready to stain with the CD8 Activation 1 cocktail and concentration-matched Isotype Control cocktail.

CD8+ T cells, or cytotoxic T lymphocytes, are immune cells that are responsible for the detection and clearance of virally or bacterically infected host cells. Upon recognition of antigen displayed on the surface of infected cells, activated CD8+ T cells upregulate markers of early activation such as CD69 and FAS ligand, followed by the release of granules containing granzymes and perforin as well as secretion of cytokines such as IFN gamma and TNF alpha. Granzymes and perforin act in concert to directly lyse infected target cells, while cytokines have paracrine effects including the recruitment and activation of macrophages, lymphocytes, and PMNs. IFN gamma is important for the upregulation of MHC class I and II on nearby cells as well as promoting Th1 cell differentiation.

Reactivity/Species
Human

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit (Invitrogen™)

The CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit enables the flow cytometric detection of activated caspase-3 and caspase-7 in apoptotic cells. The kit includes the novel fluorogenic substrate CellEvent™ Caspase-3/7 Green Detection Reagent, as well as SYTOX™ AADvanced™ Dead Cell Stain.

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

• Caspase-3/7 specific—includes the recognition sequence for activated caspase-3 and caspase-7
• Easy identification—clearly identify live, dead, and apoptotic cell populations
• Quick analysis—washing and fixation is not required
• Multicolor compatibility—combine with other dyes excitable by the 488 nm laser or other lasers

CellEvent Caspase-3/7 Green Detection Reagent is a cell-permeant reagent that consists of a four-amino acid peptide (DEVD, a caspase 3/7 recognition sequence) conjugated to a nucleic acid-binding dye. During apoptosis, caspase-3 and caspase-7 proteins are activated and cleave the peptide. Cleavage of the peptide allows binding of DNA by the reagent, thereby labeling the apoptotic cells with a bright, fluorogenic signal that has absorption/emission maxima of ~502/530 nm. When used together with the SYTOX AADvanced Dead Cell Stain, apoptotic cells can be easily discriminated from live and necrotic cells.

Because no single parameter defines apoptosis in all systems, we strongly suggest using a combination of different measurements for reliable detection of apoptosis. For more information, see Apoptosis Reagents in Flow Cytometry.

CyQUANT™ Direct Cell Proliferation Assay (Invitrogen™)

Experience a more convenient workflow for your high-throughput cytotoxicity and proliferation screening projects with the CyQUANT® Direct Cell Proliferation Assay–no washes, cell lysis, temperature equilibrations, or radioactivity required. DNA-based assays have been shown to be among the most sensitive cell health indicators and data concordance with metabolically based assays is excellent. The CyQUANT® Direct assay is a highly sensitive and robust method to assess cell growth, viability, or compound toxicity in a range of applications, from high throughput screening to bioproduction.

Features of the CyQUANT® Direct Cell Proliferation Assay include:

• More convenient workflow for high-throughput screening–no washes, cell lysis, temperature equilibrations, or radioactivity required
• Rapid protocol, high sensitivity and dynamic range
• Measures independent of metabolic state of cells on standard fluorescent plate readers
• Multiplex assays using a spectrally distinct fluorescent or a luminescent readout

Rapid and convenient, with a large dynamic range
The CyQUANT® Direct assay is a fluorescence-based proliferation and cytotoxicity assay for microplate readers with a linear detection range from less than 100 to 20,000 cells per well in most cell types. The no-wash, homogenous format and fast add-mix-read protocol makes the CyQUANT® Direct assay ideal for HTS applications. The assay can be completed in one hour, with no washes, no cell lysis, or temperature equilibrations required. The signal is stable for several hours, affording additional work flow convenience.

How it works
The CyQUANT® Direct assay is based on a cell-permeant DNA-binding dye in combination with a masking dye reagent. Cellular DNA is highly regulated and estimates of cell numbers based on DNA are very accurate. The masking dye blocks staining of dead cells or cells with compromised cell membranes resulting in only healthy cells being stained. The CyQUANT® Direct assay, therefore, measures proliferation as well as membrane integrity, another measure of cell health. Users of metabolically based cell health assays can readily compare historical data with results from the CyQUANT® Direct assay, as concordance assays based on metabolism or energy (ATP) has been shown to be excellent. Furthermore, it is possible to multiplex with spectrally distinct fluorescent probes or luminescence assay kits because cells are not lysed. Furthermore, the fluorescent signal allows the user to switch between HTS and HCS plate readers if multiplex assays require different platforms.

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

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.

eZKine™ 4-Color Compensation Control Kit (Invitrogen™)

This eZKine™ 4-Color Compensation Control Kit is designed for compensation setup with eZKine™ Whole Blood Intracellular Cytokine Kits. It contains the necessary components to create single-color compensation controls for use with eZKine™ cocktails, including OneComp eBeads and 4 vials of antibody representing each of the 4 fluorochromes used in eZKine™ cocktails. OneComp eBeads can be stained with each of the 4 fluorochrome-conjuated antibodies to set up single-color compensation control tubes.

Reactivity/Species
Human

Reported Application
Flow Cytometric Analysis

LIVE/DEAD™ BacLight™ Bacterial Viability Kit, for microscopy (Invitrogen™)

The LIVE/DEAD BacLight Bacterial Viability Kit *for microscopy* is a convenient and easy-to-use kit for monitoring the viability of bacterial populations as a function of the membrane integrity of the cell. Cells with a compromised membrane that are considered to be dead or dying will stain red, whereas cells with an intact membrane will stain green.

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.

Contains Calcein, AM, cell permeant dye as live cell indicator and BOBO-3 Iodide as dead cell indicator.

See other Ready Probes ready-to-use imaging reagents and accessories ›

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

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.

Arcturus™ Paradise™ PLUS 1.5 Round Kit, 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.

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.

Vybrant™ Multidrug Resistance Assay Kit (Invitrogen™)

The Vybrant® Multidrug Resistance Assay Kit provides a rapid and simple method for large-scale screening of MDR inhibitors. This assay uses the fluorogenic dye calcein, AM as a substrate for efflux activity of Pgp. Calcein AM is nonfluorescent, highly lipid-soluble dye that can rapidly penetrate the plasma membrane of normal cells. Once inside the cell, ester bonds are cleaved by endogenous esterase, transforming calcein AM into the hydrophilic and intensely fluorescent dye, calcein. Multidrug resistant (MDR) cells expressing high levels of P-glycoprotein (Pgp) rapidly extrude nonfluorescent calcein AM from the plasma membrane, reducing the accumulation of fluorescent calcein relative to normal cells.

eBioscience™ BrdU Staining Kit for Flow Cytometry APC (Invitrogen™)

The eBioscience BrdU Staining Kit for Flow Cytometry contains the necessary reagents and buffers for identifying and examining proliferating cells of mammalian species by flow cytometric analysis. Cycling cells are incubated with 5-bromo-2’-deoxyuridine (BrdU), a synthetic analog of thymidine which incorporates into newly synthesized genomic DNA during the S-phase of mitosis. Following DNA denaturation, the cells are stained for BrdU incorporation along with any other cell surface and/or intracellular targets of interest.

This kit is optimized to achieve brighter staining compared to traditional protocols.

Reactivity/Species
Bovine, Canine, Cow, Human, Monkey, Mouse, Non-human primate, Rat, Rhesus

Conjugate
APC

Laser
Red Laser

Emit
660 nm

Excite
633 - 647 nm

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

eBioscience™ Annexin V Apoptosis Detection Kit eFluor™ 450 (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).

Reported Application
Flow Cytometric Analysis