Flow Cytometry Reagents

Modern developments in flow cytometry, combined with the ongoing emergence of novel fluorescent labels have enabled an unprecedented level of analysis in cell biology, immunology, and microbiology, as well as, cancer and clinical research studies. With these tools, distinct populations of cells can be screened and characterized on a cell-by-cell basis for specific, biologically-significant traits, including cell surface markers and secreted markers, intracellular targets, enzyme activity, nucleic acid content, apoptosis and much more. AAT Bioquest provides a comprehensive portfolio of flow cytometry reagents ranging from fluorescent labels and CD antibody conjugates to fixable viability dyes, cell proliferation dyes and tools to monitor apoptosis.

Super Bright Fluorescent Dyes, Phycobiliproteins and Tandem Dyes for Multicolor Flow Cytometry

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Fluorescent dyes conjugated to antibodies or proteins enable biological and biochemical properties to be measured in a wide range of flow cytometric applications. Across most experimental conditions, phycobiliproteins such as PE and APC are among the brightest fluorophores available for flow cytometry. While novel mFluor™ dyes offer users greater photostability, superior brightness and exceptionally long Stoke's Shifts for multiparametric analysis.

To label primary antibodies or proteins for flow cytometry, AAT Bioquest offers a wide selection of antibody labeling kits. Our ReadiLink™ Rapid mFluor™ Antibody Labeling Kits and Buccutite™ PE, APC and Tandem dye Antibody Labeling Kits, enables researchers to effortlessly label and recover 100% of their antibodies without a purification step. Produce stable covalently-labeled conjugates for FACS, immunophenotyping, multiplex flow cytometry and more.

Labeling Kit	CD4(SK3)- Conjugate	Stain Index
Buccutite™ Kit	CD4-PE Conjugate	55
Lightning-Link® Kit	CD4-PE Conjugate	28

Cell Proliferation Reagents for Flow Cytometry

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Cell proliferation assays and reagents are important tools used in cell growth and differentiation studies to monitor the health of cells within a heterogeneous population. They have been used extensively in pharmaceutical drug development to examine compound toxicity and inhibition of tumor cell growth, and have proved to be a successful diagnostic tool to detect the onset of several cancers. In flow cytometry, several approaches can be used to measure proliferation; these include dye dilution assays and DNA synthesis measurements through the incorporation of Bromodeoxyuridine (BrdU).

Dye dilution assays for cell proliferation

CFSE and CytoTell™ proliferation dyes can be used in flow cytometry to examine cell proliferation in living cells. As cells divide, the fluorescent dye is uniformly distributed between daughter cells, resulting in the progressive halving of fluorescence intensity with each successive generation. These proliferation dyes can be used to track cell proliferation long-term both in vitro and in vivo for several generations.

CFSE (Carboxyfluorescein diacetate, succinimidyl ester) is a cell-permeable green fluorescent proliferation indicator that emits a fluorescence signal at 517 nm when excited at 498 nm. This dye passively diffuses across uncompromised cell membranes, whereby enzyme reactions with intracellular esterases cleave acetate groups of CFSE to yield an amine-reactive carboxyfluorescein, SE that covalently binds to proteins within the cell. While CFSE still remains widely used as a green fluorescent proliferation indicator excited at 498 nm it is, however, not without caveats. CFSE is highly toxic to cells, susceptible to dye leakage from the cell and is known to spillover into the PE and PE-Texas Red channels.

High-performance CFSE upgrades for cell proliferation

CytoTell™ are a series of cell-permeable fluorecent tracers designed to monitor cell proliferation in living cells without any of the drawbacks associated with CFSE. Upon passive diffusion across cell membranes, CytoTell™ dyes are hydrolyzed by intracellular esterases to yield highly-fluorescent amine-reactive or thiol-reactive dyes that covalently bind to amine or thiol groups on intracellular proteins. Cytotoxicty is minimal and the dyes are well-retained within cells through several generations of division, up to 9 generations can be visualized using CytoTell™ UltraGreen. CytoTell™ dyes are available in 7 fluorescent stains offering greater flexibilty in multicolor analysis, and may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers.

Fixable Viability Dyes for Flow Cytometry

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The ability to discriminate and exclude dead cells from live cells enables cleaner separation and identification of cell populations. It greatly enhances assay accuracy, and eliminates undesirable artifacts and false positives caused by the nonspecific binding of various reagents to dead cells. To distinguish live and dead cell populations from flow cytometry readouts AAT Bioquest offers a broad range of Cell Meter™ Fixable Viability dyes and Live or Dead™ Fixable Dead Cell Staining Kits. Labeling is stable, robust and compatible with fixatives and permeabilization buffers allowing for further analysis of intracellular targets.

Cell Meter™ fixable viability dyes

Cell Meter™ fixable viability dyes are a family of membrane-impermeant viability dyes designed to distinguish live and dead cells during flow cytometry. These dyes readily permeate dead cells with compromised membrane integrity and bind irreversibly to amine- and thiol-containing proteins and other intracellular components making it easy to exclude them from analysis. Because staining is robust and stable, cells labeled with Cell Meter™ fixable viability dyes can be fixed, permeabilized and probed for intracellular immunophenotyping.

The Cell Meter™ fixable viability dyes are available in 9 fluorescent stains to provide users greater flexibility in multicolor panel design. Dyes included in this series are excited by major excitation sources including the 405, 488, 633 and 647 nm laser lines, and emit in common channels.

Live or Dead™ Fixable Dead Cell Staining Kits

Live or Dead™ Fixable Dead Cell Staining Kits employ membrane-impermeant amine-reactive dyes to differentiate live and dead cells during flow cytometry. The dyes provided in each kit can readily permeate compromised membranes of dead cells and covalently bind to both intracellular and extracellular amine-containing proteins resulting in intense fluorescence. While on live cells, the fluorescence signal is negligible as cell surface amine-groups are only avaiable to react with the dye. The difference in signal intensity between live and dead cell populations is signifcant, making it easy to exclude dead cells from analysis. Cells labeled using Live or Dead™ Fixable Dead Cell Staining Kits can be fixed, permeabilized and probed for intracellular immunophenotyping. AAT Bioquest offers 10 Live or Dead™ Fixable Dead Cell Staining Kits designed to for efficient excitation by major exciation sources including the 350, 405, 488, 532, 561, 633 and 647 nm laser lines, and emit in common channels.

Live and Fixed Cell Cycle Assays for Flow Cytometry

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Analyzing changes in DNA content during flow cytometry is a powerful method commonly used to identify cells in different stages of the cell cycle. This technique utilizes fluorescent DNA binding dyes, which bind to DNA stoichiometrically, generating a fluorescence signal that is directly proportional to the amount of DNA present in the cell. This way when the DNA density increases as cells progress from the G1 phase to the G2 phase of the cell cycle, cells will take up proportionally until the cell have doubled their DNA content. As a result cells in the G2 phase will fluoresce twice as brightly as cells in the G1 phase.



AAT Bioquest offers a selection of 4 cell cycle assays, 3 of which are designed for live cell analysis and 1 for fixed cells. The Cell Meter™ Fluorimetric Live Cell Cycle Assay Kits utilize membrane-permeant DNA binding dyes to monitor cell cycle progression and proliferation, and can be used to investigate the kinetic and temporal aspects of the cell cycle. After staining cells can be fixed and permeabilized. The Cell Meter™ Fluorimetric Fixed Cell Cycle Assay Kit utilizes our proprietary membrane-impermeant DNA binding dye Nuclear Red™ CCS1. Prior to staining, cells must be fixed or permeabilized to all the dye to enter the cell. Each kit includes a robust staining protocol and all the necessary components for 200 tests. AAT Bioquest also offers a large selection of stand-alone DNA binding dyes for cell cycle analysis, including propidium iodide, 7-AAD, DAPI, Hoechst 33342, 33258, S769121, 34580, and TWO-PRO™ 1 and TWO-PRO™ 3.

Apoptosis Assay for Flow Cytometry

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Flow cytometry is one of the most versatile and high-throughput applications for studying the complexities of apoptosis. Not only does it enable researchers to analyze cells individually from complex populations, but the multiparametric nature of flow cytometry allows for several apoptotic indicators and markers to be examined simultaneoulsy. With our broad selection of flow cytometric apoptosis assays investigate changes in plasma membrane conformation, loss of mitochondrial membrane potential, activation of caspases, nuclear condensation or utilize a combination of techniques for multiparametric apoptosis analysis.

Annexin V assays for the detection of apoptosis by flow cytometry

Annexin V conjugates are routinely used to investigate the externalization of phosphatidylserine (PS) residues, a key characteristic in early-stage apoptosis. As cells initiate programmed cell death, they lose plasma membrane asymmetry and PS residues translocate from the inner to the outer leaflet of the plasma membrane. Exposure of PS residues on the cell surface can be detected by its affinity for annexin V, and when fluorescently labeled annexin V conjugates can be used to identify apoptotic cells in flow cytometry.

AAT Bioquest Cell Meter™ Annexin V Binding Apoptosis Assay Kits offer a quick and reliable method for measuring apoptotic cells based on the externalization of PS residues. These kits utilize a highly fluorescent annexin V conjugate and a membrane-impermeant DNA stain to differentiate between live, dead and apoptotic cells. In this way, cells that are viable show little or no fluorescence, cells undergoing early apoptosis show annexin V conjugate fluorescence and dead cells show a double labeling of the membrane-impermeant DNA stain and annexin V conjugate. These kits can be paired with other reagents for multi-parametric study of cell viability and apoptosis.

Key features of Cell Meter™ Annexin V Bindinng Apoptosis Assays

• High Sensitivity - annexin V labeled with iFluor® or mFluor™ dyes produce significantly brighter and more photostable signals than Alexa Fluor^® and other spectrally similar annexin V conjugates
• Flexibility - wide selection of stand-alone annexin V conjugates or easy-to-use assay kits to match any instrument set-up
• Convenient assay design - streamlined protocol that can be completed in 30 to 60 minutes

1. For Cat No. 22825, 22826 and 22827 use Nuclear Blue™ DCS1 as a dead cell stain.

Mitochondria membrane potential assays for flow cytometry

Loss of mitochondrial membrane potential is a distinctive feature of the early stages of apoptosis. In flow cytometry, membrane-permeant cationic probes can be used to identify changes in the mitochondrial membrane potential in real-time and discriminate apoptotic cells from live cell populations. AAT Bioquest offers a wide selection of reagents and dynamic assays designed to monitor mitochondrial membrane potential, these include ratiometric indicators such as JC-10™, single-emission indicators, and Cell Meter™ Mitochondrion Membrane Potential Assay Kits optimized for flow cytometry applications.

JC-10™

AAT Bioquest JC-10™ is a novel ratiometric indicator designed to monitor mitochondrial membrane potential with greater accuracy and sensitivity than its predecessor JC-1. Similar to JC-1, JC-10™ exhibits potential dependent accumulation. In healthy cells, JC-10™ selectively accumulates in mitochondria generating orange J-aggregates that exhibit a broad excitation spectrum and emission maximum at 590 nm. Conversely, in apoptotic and necrotic cells with low mitochondrial membrane potential, JC-10™ diffuses out of the mitochondria and JC-10™ monomers are generated, resulting in a shift to green emission (525 nm). JC-10™ allows both qualitative and quantitative visualization of mitochondrial membrane potential changes, considering the shift from orange to green fluorescence emission and the fluorescence intensity ratio, respectively. JC-10™ dye is available as a standalone reagent or in the Cell Meter™ JC-10 Mitochondrion Membrane Potential Assay Kit *Optimized for Flow Cytometry Assays*

Key features of JC-10™ and the Cell Meter™ JC-10™ assay kit

• Easy-to-Use: JC-10™ does not percipitate when diluted into aqueous buffers, eliminating artifacts.
• Robust: JC-10™ has smaller assay deviations due to its enhanced solubility in aqueous media and higher sensitivity.
• Enhanced Signal: JC-10™ has a higher signal-to-background ratio than JC-1.
• Enhanced Sensitivity: JC-10™ has the ability to detect subtle changes in ΔΨm loss better than JC-1 in all tested cell lines.

Single-emission mitochondrial membrane potential dyes

Other reagents and assays that can be used to dynamically monitor changes in mitochondria membrane potential include TMRE, TMRM and the Cell Meter™ Mitochondrion Membrane Potential Assay Kits available in orange, red and near-infrared fluorsecence. In healthy cells with functioning mitochondria, these membrane-permeant dyes are readily sequestered by active mitochondria and fluoresce brightly. When mitochondria membrane potential collapses, accumulation ceases and the dyes are released throughout the cytosol resulting in loss of fluorescence.