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MANT-ATP (2'-(or-3')-O-(N-Methylanthraniloyl) Adenosine 5'-Triphosphate, Trisodium Salt) Invitrogen™

The nucleotide analog MANT ATP is modified on the ribose moiety. The compact mature of the MANT fluorophore and its attachment position results in nucleotide analogs that induce minimal perturbation of nucleotide-protein interactions. Because MANT fluorescence is sensitive to the environment of the fluorophore, nucleotide-protein interactions may be directly detectable. MANT nucleotides are valuable probes of the structure and enzymatic activity of nucleotide-binding proteins.

Tango™ PTAFR-bla U2OS Cells Thermo Scientific™

The Tango™ PTAFR-bla U2OS cells contain the human Platelet Activating Factor Receptor (PTAFR) linked to a TEV protease site and a Gal4-VP16 transcription factor stably integrated into the Tango™ GPCR-bla U2OS parental cell line. This parental cell line stably expresses a beta-arrestin/TEV protease fusion protein and the beta-lactamase (bla) reporter gene under the control of a UAS response element.

The Tango™ PTAFR-bla U2OS cells are functionally validated for Z' and EC50 concentrations of β-Acetyl-γ-O-hexadecyl-L-α-phosphatidylcholine. In addition, Tango™ PTAFR-bla U2OS cells have been tested for assay performance under variable conditions. These data are found in the Validation & Assay Performance Summary.

For Academic pricing please login or contact us at discoverysciences@invitrogen.com.

Dead Cell Apoptosis Kit with Annexin V APC and SYTOX™ Green, for flow cytometry Invitrogen™

This flow cytometry product detects the externalization of phosphatidylserine in apoptotic cells using recombinant annexin V conjugated to red laser-excited allophycocyanin, and dead cells using SYTOX™ Green nucleic acid stain. Apoptotic cells are detected by annexin V binding to externalized phosphatidylserine, and late apoptotic and necrotic cells have compromised membranes that permit SYTOX™ Green stain access to cellular nucleic acids. After exposing a cell population to APC annexin V and SYTOX™ Green stain, live cells show little or no fluorescence, apoptotic cells show red fluorescence and very little green fluorescence, and late apoptotic cells show a higher level of red and orange fluorescence. These populations can easily be distinguished using a flow cytometer that has both 488 nm and 633 nm excitation sources (an argon-ion laser and a HeNe laser).

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

CellSensor™ NFAT-bla CHO-K1 Cell Line Invitrogen™

The G protein-coupled receptor (GPCR) superfamily is comprised of an estimated 600-1,000 members and is the largest known class of molecular targets with proven therapeutic value. GPCRs are instrumental in the transmission of a wide range of chemical messages from the extracellular environment to the interior of the cell. GPCRs are involved in a wide range of disorders, including allergies, cardiovascular dysfunction, depression, obesity, cancer, pain, diabetes, and various central nervous system disorders. GPCR signaling is mediated by trimeric G-proteins containing α, β, and γ subunits and can be categorized into signaling classes based upon subunit composition. G alpha, G alpha s, and G alpha i/o proteins mediate intracellular signaling through activation of signaling pathways leading to distinct physiological endpoints. Activation of G alpha s and G alpha i/o coupled receptors leads to stimulation or inhibition of adenylate cyclase, respectively; activation of G alpha q coupled receptors results in stimulation of phospholipase C. GPCR signaling through these distinct pathways can be monitored by activation of specific transcriptional response elements placed upstream of a reporter gene. Invitrogen has developed GeneBLAzer® Master Cell Lines employing the beta-lactamase reporter system, which can be used to screen for both agonists and antagonists of GPCRs coupling to each of the above signaling pathways. These cell-based assays make use of a membrane permeant fluorescent substrate (CCF4-AM or CCF2-AM), enabling a ratiometric readout that reduces errors due to sample variability, leads to excellent Z'-factor values, and allows for assay miniaturization. GeneBLAzer® Master Cell Lines with either NFAT or CRE response elements are available in Jurkat, CHO-K1, or FreeStyle™ 293F cell backgrounds (six cell lines in total) for the development of assays in suspension or adherent cell format. Stable cell lines employing an NFAT response element upstream of the beta-lactamase gene are designed for the study of Gq coupled receptors. Stable cell lines employing the cAMP response element (CRE) upstream of the beta-lactamase gene are designed for the study of Gs coupled receptors. FreeStyle™ 293F cells are HEK 293 cells that have been adapted for growth in suspension in serum-free media. Alternatively, these cells may be grown in the presence of serum for use in the adherent format. Academic and non-profit customers, please inquire for special pricing.

ViewRNA™ ISH Cell Assay Kit Invitrogen™

The ViewRNA™ ISH Cell Assay is a direct fluorescence RNA in situ hybridization method that enables the simultaneous detection of one to four RNA targets (either mRNA or non-coding RNA) at single copy sensitivity and single cell resolution using fluorescence microscopes or high-content imagers. Unlike traditional FISH techniques which are generally limited by high background and low sensitivity, the assay uses proprietary chemistry for the target specific probe sets (RNA FISH probes) and branch DNA signal amplification (bDNA) for detection of specific signal. The fluorescence RNA in situ hybridization assay has four main steps: sample preparation, target hybridization, signal amplification, and detection. This ViewRNA ISH assay kit can detect up to three RNA but can be combined with the ViewRNA™ 740 Module to add the fourth probe.

Required additional components:
10X PBS (cat. no. QVC0508)
ViewRNA™ - Detergent Solution QC (cat. No. QVC0509)
ViewRNA™ - Wash Buffer Set (cat. no. QG0507)
ViewRNA™ ISH Cell Accessory Kit (cat. no. QVC0700) intended to provide many of the required components, not supplied in the reagent kit, in order to perform the assay.
ViewRNA™ Probe Sets are not included in the assay and are designed for use with the ViewRNA ISH Cell Assays. Visit our website to view a complete listing of over 6,500 synthesized Probe Sets. By request, new Probe Sets can be designed and synthesized in less than two weeks with no additional costs.

To add the 4th RNA probe:
ViewRNA™ ISH Cell 740 Module (cat. no. QVC0200) designed to be used in conjunction with ViewRNA™ ISH Cell Assay and allows analysis of an additional RNA target in the 740 channel (AlexaFluor 750) See the package insert for a complete list of materials provided in the kit.

Reported Applications
Microscopy

7-AAD (7-Aminoactinomycin D) Invitrogen™

7-aminoactinomycin D (7-AAD) is a fluorescent intercalator that undergoes a spectral shift upon association with DNA. 7-AAD/DNA complexes can be excited by the 488 nm laser and has an emission maxima of 647 nm, making this nucleic acid stain useful for multicolor fluorescence microscopy and flow cytometry. 7-AAD appears to be generally excluded from live cells, but can be used with cells that have been fixed and permeabilized. 7-AAD has been used for cell cycle analysis by flow cytometry. 7-AAD also binds selectively to GC regions of DNA yielding a distinct banding pattern in polytene chromosomes and chromatin for use in chromosome banding studies.

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

GeneBLAzer™ Bradykinin-B2-NFAT-bla CHO-K1 Cells Thermo Scientific™

GeneBLAzer® Bradykinin-B2-NFAT-bla CHO-K1 cells contain the human Bradykinin Receptor B2 (B2) stably integrated into the CellSensor® CRE-bla CHO-K1 cell line. CellSensor® NFAT-bla CHO-K1 cells (Cat. no. K1534) contain a beta-lactamase reporter gene under control of the Cyclic AMP Response Element (CRE) response element.

The Bradykinin-B2-NFAT-bla CHO-K1 cells are functionally validated for Z' and EC50 concentrations of Bradykinin. In addition, GeneBLAzer® Bradykinin-B2-NFAT-bla CHO-K1 cells have been tested for assay performance under variable conditions. These data are found in the Validation & Assay Performance Summary.

For Academic pricing please login or contact us at discoverysciences@invitrogen.com.

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.

Metabolic Activity Dead Cell Apoptosis Kit with C12 Resazurin, Annexin V APC, and SYTOX™ Green, for flow cytometry Invitrogen™

This flow cytometry product provides a three-color fluorescence assay that distinguishes live, apoptotic, and late apoptotic cells from one another. These populations can easily be distinguished using a flow cytometer that has both 488 nm and 633 nm excitation sources (an argon-ion laser and a HeNe laser) and the following reagents Annexin V to detect phosphatidylserine, C12 resazurin for cell metabolism, and SYTOX™ Green nucleic acid stain for compromised membranes.

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

FluxOR™ Potassium Ion Channel Assay Invitrogen™

K+ channel specific—measure ion flux in both voltage- and ligand-gated potassium channels
Fast—perform screens in high-throughput mode with reproducible results and excellent S/N without quenching dye
Pharmacologically relevant—known blockers show dose-dependent inhibition in a large signal window

The FluxOR™ Potassium Ion Channel Assay is an optically based, homogenous assay for high throughput screening (HTS) measurements of potassium ion channel and transporter activities. The homogenous assay is based on the permeability of potassium channels to thallium I. When potassium channels are opened by a stimulus, thallium influx from the external medium is detected with a highly sensitive indicator dye. The fluorogenic signal quantitatively reflects the activity of ion channels and transporters that are permeant to thallium, including hERG, Kir2.1, and other pharmacologically important potassium channels. Fluorescence reported in the FluxOR™ system thus becomes a surrogate indicator of activity for any ion channel or transporter that is permeable to thallium.

The FluxOR™ Potassium Ion Channel Assay enables rapid and robust high-throughput screening (HTS) of potassium channel targets in a novel equilibrium measurement, reproducibly giving IC50 values that are predictive of block or modulation in lower-throughput platforms. The FluxOR™ dye is sensitive enough that low mM levels of extracellular thallium give large signals in high-throughput mode. For most applications, FluxOR™ dye is dissolved in physiological HBSS buffer for loading into cells, assisted by our proprietary Powerload™ formulation (Catalog number P10020).

The FluxOR™ Potassium Ion Channel Assay provides a concentrated thallium solution and all necessary buffers, allowing maximum target flexibility and ease of operation in a homogenous format that has been demonstrated for use with cells stably expressing hERG, as well as our BacMam-hERG delivery and expression reagent (Catalog number B10019 and B10033). Optional for use with this kit is the hERG potassium channel cDNA engineered into Invitrogen’s BacMam delivery and expression system. The combination of a bright proprietary fluorescent potassium sensor dye and the hERG potassium channel gene delivered by BacMam affords excellent assay design flexibility and superior sensitivity in detecting potassium channel activity in biologically relevant systems at physiological conditions without the need for quenchers.

CellSensor™ CRE-bla CHO K1 Cell Line Thermo Scientific™

The CellSensor® CRE-bla CHO-K1 cell line contains a beta-lactamase reporter gene under control of a cAMP response element (CRE) stably integrated into CHO-K1 cells. This cell line can be used as a parental cell line to build specific G-protein coupled receptor (GPCR) assays after transfection of additional genes of interest or to detect changes in intracellular cAMP levels. CRE-bla CHO-K1 cells have been shown to be responsive to forskolin stimulation. Academic and non-profit customers, please inquire for special pricing.

PrestoBlue™ Cell Viability Reagent Invitrogen™

Get high quality cell viability results and instant time savings with PrestoBlue® Cell Viability Reagent. PrestoBlue® is a ready to use cell permeable resazurin-based solution that functions as a cell viability indicator by using the reducing power of living cells to quantitatively measure the proliferation of cells. When added to cells, the PrestoBlue® reagent is modified by the reducing environment of the viable cell and turns red in color, becoming highly fluorescent. This color change can be detected using fluorescence or absorbance measurements.

The PrestoBlue® Cell Viability Reagent allows you to:

• Save time with an incubation step as short as 10 minutes.
• Obtain high quality results with a large dynamic range
• Experience convenience with the add and read homogenous format
• Monitor living cells and perform downstream functional assays

10-minute incubations
The PrestoBlue® Cell Viability Reagent is the only live-cell viability reagent on the market able to provide robust data typically in as little as 10 minutes following reagent addition. All other resazurin-based cell viability reagents on the market require a one to four hour incubation with the reagent prior to obtaining data.

High quality results
Based on the cell type and incubation time, PrestoBlue® Cell Viability Reagent can detect as few as 12 cells per well in a 384 well plate. After ten minutes of incubation the PrestoBlue® Reagent can linearly detect cells down to 98 cells⁄well with a Z’ value of greater than 0.5.

Homogenous assay format
The PrestoBlue® Cell Viability Reagent is an addition-only reagent. Without the cell lysis or solubilization steps required by other assays on the market, hands-on time is minimized.

Live cell assays
The PrestoBlue® Cell Viability Reagent does not require cell lysis. Cells may be incubated for up to 24 hours in the presence of PrestoBlue® Cell Viability Product. Simply remove the reagent from the cells and replace it with growth medium for further proliferation.

Fluo-4, AM, cell permeant Invitrogen™

Labeled calcium indicators are molecules that exhibit an increase in fluorescence upon binding Ca2+. Fluo-3 has been used to image the spatial dynamics of Ca2+ signaling, in flow cytometry experiments involving photoactivation of caged chelators, second messengers, and neurotransmitters, and for cell-based pharmacological screening. Fluo-4 is an analog of fluo-3 with the two chlorine substituents replaced by fluorines, which results in increased fluorescence excitation at 488 nm and consequently higher fluorescence signal levels. Cells may be loaded with the AM ester forms of these calcium indicators by adding the dissolved indicator directly to dishes containing cultured cells. These indicators are useful for fluorescence and confocal microscopy, flow cytometry, and microplate screening applications.

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

Calcium Indicator (AM Ester) Specifications:
• Label (Ex/Em of Ca2+–bound form): Fluo-4 (494/506 nm)
• Fluorescence intensity increase upon binding Ca2+: >100 fold
• Kd for Ca2+ in buffer: ~335 nM
• Exhibit fluorescence increase upon binding Ca2+ with little shift in wavelength


Using TPEN to Control Heavy Metal Cations
In addition, BAPTA-based indicators such as these bind various heavy metal cations (e.g., Mn2+, Zn2+, Pb2+) with substantially higher affinity than Ca2+. Perturbations to calcium measurements caused by presence of these ions can be controlled using the heavy metal-selective chelator TPEN.

More Choices for Fluorescent Calcium Indicators
We offer a large selection of Molecular Probes® calcium indicators for use in various experimental scenarios. For more information, review Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3 in the Molecular Probes® Handbook.

For UV-excitable Ca2+ indicators, protein-based Ca2+ indicators, conjugates of Ca2+ indicators, and for fluorescence-based indicators of other metal ions (i.e., Mg2+, Zn2+) review Indicators for Ca2+, Mg2+, Zn2+ and Other Metal Ions—Chapter 19 in the Molecular Probes® Handbook.

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

alamarBlue™ Cell Viability Reagent Invitrogen™

alamarBlue Cell Viability Reagent is a ready-to-use resazurin-based solution that functions as a cell health indicator by using the reducing power of living cells to quantitatively measure viability. Resazurin, the active ingredient of alamarBlue reagent, is a non-toxic, cell-permeable compound that is blue in color and virtually non-fluorescent. Upon entering living cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent. Changes in viability can be easily detected using either an absorbance- or fluorescence-based plate reader. alamarBlue Cell Viability Reagent has broad applicability and can be used with various human and animal cell lines, bacteria, plant, and fungi.

Features include:
• Robust and reliable performance—results in a large, highly reproducible dynamic range
• Highly sensitive reagent with a linear response—detects as few as 50 cells per well
• Convenient add-and-read format—no mixing, no washing, no cell lysis
• Compatible with either fluorescence- or absorbance-based instrumentation
• Measures viability from many diverse cell types—including mammalian cells, bacteria, plant, and fungi

Cell health can be monitored by numerous methods. Plasma membrane integrity, DNA synthesis, DNA content, enzyme activity, presence of ATP, and cellular reducing conditions are known indicators of cell viability and death. alamarBlue Cell Viability Reagent is an indigo-colored, non-toxic reagent that detects metabolically active cells and is used for the quantitative analysis of cell viability and proliferation. alamarBlue reagent has broad applicability and can be used with various human and animal cell lines, bacteria, plant, and fungi.

When added to cells, alamarBlue Cell Viability Reagent is modified by the reducing environment of viable cells and turns red in color and becomes highly fluorescent. This color change and increased fluorescence can be detected using absorbance (detected at 570 and 600 nm) or fluorescence (using an excitation between 530–560 and an emission at 590 nm). To assay for viability, simply add the pre-mixed alamarBlue reagent to cells in complete media (no wash or cell lysis steps required), incubate for one to four hours, and read using either an absorbance- or fluorescence-based plate reader. If necessary, longer incubation times may be used for greater sensitivity without compromising cell health.

The homogeneous, add-and-read assay format of the alamarBlue Cell Viability Reagent is fast, convenient, and readily amenable to automation and high-throughput assays. The color change and fluorescence increase resulting from cell viability changes allow the detection of alamarBlue reagent by either an absorbance- or fluorescence-based instrumentation.

TNP-ATP (2'-(or-3')-O-(Trinitrophenyl) Adenosine 5'-Triphosphate, Trisodium Salt) Invitrogen™

The nucleotide analog TNP-ATP is modified on the ribose moiety and is essentially non-fluorescent in water. The TNP nucleotides undergo an equilibrium transition to a semiquinoid structure that has relatively long-wavelength spectral properties; this form is only fluorescent when bound to the nucleotide-binding site of some proteins.
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