Parameters that define cell viability, such as plasma membrane integrity, mitochondrial membrane potential (ΔΨm), and intracellular esterase activity, can collectively be used to differentiate live and dead cell populations, determine cell vitality, and screen for compound cytotoxicity. These parameters form the basis for strategies such as our Live or Dead™ Cell Viability Assays, which combine two fluorescent reagents - a live cell esterase substrate and a cell-impermeable DNA binding dye - to yield two-color discrimination of the live and dead cells within a sample population. Live or Dead™ assays can be used to examine eukaryotic cells and bacteria by fluorescence microscopy, flow cytometry, or fluorometry.

AAT Bioquest Live or Dead™ Cell Viability Assay Kits are based on the simultaneous determination of live and dead cells using two fluorescent probes. Live cells are identified using either a calcein acetoxymethyl (AM) derivative or a mitochondrial membrane potential probe, while dead cells are labeled using a cell-impermeable DNA binding dye. The calcein AM derivatives are non-fluorescent esterase substrates that readily diffuses across intact cell membranes and permeate into live cells. Once inside, non-specific intracellular esterases hydrolyze the substrate into a fluorescent byproduct trapping it within the cell. The fluorescence signal emitted by the substrate is an indicator of live cells having esterase activity and an intact membrane. Alternatively, mitochondrial membrane potential probes can also be used to distinguish live cells. These membrane-permeant, cationic dyes passively diffuse across the plasma membrane and accumulate within the mitochondria of healthy cells generating significant fluorescence. In contrast, dead cells that have lost their ΔΨm will exclude the dye, and no signal will be observed. The cell-impermeable DNA binding dye, which binds selectively and with a high affinity to DNA, can only penetrate the compromised membranes of dead cells and will fluoresce significantly upon binding to nucleic acids. Healthy live cells with intact cell membranes, however, will exclude the dye from entering the cell, resulting in a negative signal. Because the determination of cell viability depends on these physical properties, cytotoxic events that do not affect these parameters may not be accurately assessed using these methods.

The Live or Dead™ Cell Viability Assay Kit *Green/Red Dual Fluorescence* is a quick and easy assay for accurately determining cell viability and cytotoxicity in eukaryotic cells. The kit features two probes - CytoCalcein™ Green and propidium iodide - that can be added simultaneously to the sample for two-color fluorescence discrimination of live and dead cells without any wash steps. CytoCalcein™ Green is a fluorogenic esterase substrate that labels live cells exhibiting esterase activity with green fluorescence. At the same time, propidium iodide is a membrane-impermeant nucleic acid stain that labels dead cells with red fluorescence. The kit can be easily adapted for high-throughput assays in a wide variety of fluorescence platforms, including fluorescence microplate readers, imaging systems, and flow cytometer, and is compatible with 3D-cell culture models, spheroids, adherent cells, non-adherent cells, and certain tissues. Compared to alternative methods for assessing cell viability, such as trypan blue exclusion assays or chromium-release assays, the Live or Dead™ Cell Viability assay is significantly safer, more cost-effective, and more sensitive at defining cytotoxic events.

The Live or Dead™ Cell Viability Assay Kit *Red/Blue Dual Fluorescence* is a convenient assay for determining cell viability and cytotoxicity based on membrane integrity and mitochondrial membrane potential activity. The kit features two probes - Cellbrite™ Red and Nuclear Blue™ DCS1 - that can be added simultaneously to the sample for two-color fluorescence determination of live and dead cells. Cellbrite™ Red stains living cells with active mitochondria red, while the membrane-impermeable DNA dye Nuclear Blue™ DCS1 stains dead cells blue. It can be easily adapted for high-throughput assays in a wide variety of fluorescence platforms, such as fluorescence microplate assays, immunocytochemistry, imaging, and flow cytometry, and is compatible with 3D-cell culture models, spheroids, adherent cells, and non-adherent cells.

The MycoLight™ Fluorescence Live/Dead Bacterial Imaging Kit provides a quick and easy method to discriminate live and dead bacteria in suspension or immobilized on filter membranes. The kit comprises two probes - MycoLight™ 520 and propidium iodide - that can be added simultaneously to the bacterial sample without any wash steps, making it amenable for high-throughput screening. MycoLight™ 520 is a membrane-permeant fluorogenic esterase substrate that passively diffuses across the plasma membrane of bacteria and stains live bacteria bright green. In contrast, propidium iodide, which can only penetrate the compromised membranes of dead bacteria, labels dead bacteria with red fluorescence. Thus, with an appropriate mixture of the MycoLight™ 520 and propidium iodide stains, live bacteria with intact cell membranes emits green fluorescence and is analyzed using standard fluorescein filter sets, whereas dead bacteria with damaged membranes give red fluorescence and are analyzed using standard TRITC filter sets.

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 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.

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 available to react with the dye. The difference in signal intensity between live and dead cell populations is significant, 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 or other multiparameter staining experiments. AAT Bioquest offers 10 Live or Dead™ Fixable Dead Cell Staining Kits designed for efficient excitation by major excitation sources, including the 350, 405, 488, 532, 561, 633, and 647 nm laser lines, and emit in common channels. These kits are exceptional tools for preserving fluorescent images of particular cells and can also be used for fluorescence microscope demonstrations.