A cell viability assay is often based on assaying ongoing cellular metabolism and enzyme activity, ie measuring factors that reflect the number of living cells in a population.
Cell viability assay methods
See below to learn more about these assay methods, or review our most popular cell viability assay kits based on MTS, resazurin, TMRE, calcein violet, and ATP luminescence.
Alternative methods of performing a cell viability assay measure it indirectly, by measuring cytotoxicity, ie the number of dead or damaged cells in a population. Learn how to run a cytotoxicity assay, by assessing cell membrane damage such as with the LDH assay or with dyes like 7-AAD.
Sometimes an apoptosis assay, such as Annexin V, TUNEL or caspase assay, or a cell proliferation / cell cycle assay, such as those using dye dilution, BrdU/EdU, or DNA staining dyes, are used to assess cell viability.
Dye reduction assays
Tetrazolium cell viability assays rely on cellular dehydrogenases to form a colored formazan product, which is measured by absorbance. Other assays use the reduction of resazurin, by electron acceptance from the mitochondrial respiratory chain, to form the fluorescent resorufin.
MTT assay ab211091: Original tetrazolium assay; still very popular. Only tetrazolium assay that needs a wash/solubilization step. Plate reader.
MTS assay ab197010: Most popular assay. More heavily used than WST-1. Plate reader.
WST-1 assays ab155902, ab65473, and ab65475: More sensitive than MTT, XTT or MTS. Plate reader.
Cell Counting Kit-8/CCK-8/ WST-8 assay ab228554: Plate reader.
XTT assay ab232856: Plate reader.
Resazurin is equivalent to the active ingredient of ThermoFisher’s alamarBlue®
Resazurin assay ab129732: Fluorometric (Ex/Em 535–560/560–615) or colorimetric. No-wash assay. Fluorescent readout enables multiplexing with other assays. Plate reader, microscope, flow cytometer.
Mitochondrial membrane potential-dependent dyes
There are several dyes available that accumulate in mitochondria due to the mitochondrial membrane potential and you can use these to identify viable cells. A loss of membrane potential and loss of staining is used to assay for apoptosis.
TMRE assay ab113852 / TMRM assay ab228569: TMRE is the most popular Abcam mitochondrial membrane dye assay. Ex/Em 549/575 nm. Washed out of mitochondria after fixation. Plate reader, microscope, flow cytometer.
JC-1 assay ab113850 / JC-10 assays ab112134 and ab112133: JC-1 (Ex/Em 530/530–570) and JC-10 (Ex/Em 590/520–570) form red aggregates at high concentrations (unaggregated dye is green). Loss of membrane potential causes loss of dye and increased green fluorescence. JC-10 is more soluble than JC-1. Washed out after fixation. Plate reader, microscope, flow cytometer.
Mitotracker Red assay: Ex/Em 579 /599. Not washed out after fixation. Plate reader, microscope, flow cytometer.
Rhodamine 123 assay: Ex/Em 507/529. Washed out after fixation. Plate reader, microscope, flow cytometer.
MitoNIR assays ab112149 and ab112150: Ex/Em 635/660. Plate reader, flow cytometer.
MitoOrange assays ab138898 and ab138899: Ex/Em 540/590. Plate reader, flow cytometer.
Calcein and similar hydrophobic dyes diffuse into cells and are cleaved by intracellular esterases in live cells. The hydrophilic fluorescent product is retained within the cell.
Calcein AM assay kit ab228556: Ex/Em 495/515 nm. Plate reader, microscope, flow cytometer.
Calcein violet AM ab176748: Ex/Em 405/460 nm. Plate reader, microscope, flow cytometer.
Esterase-cleaved blue assay ab112120: Ex/Em 360/450 nm. Plate reader.
Esterase-cleaved green assay ab112122: Ex/Em 490/520 nm. Plate reader, microscope.
Esterase-cleaved near IR assay ab112123: Ex/Em 633/660 nm. Plate reader, microscope.
Most assays use a cell membrane permeabilization agent to release ATP; light is produced using ATP-dependent luciferase. Other ATP assays use the ATP-dependent phosphorylation of glycerol (or other substrates).
Luminescence ATP assay ab113849: No-wash assay. Luminometric plate reader.
Luminescence ADP/ATP assay ab65313: No-wash assay. After ATP analysis, ADP is converted to ATP for detection. Luminometric plate reader.
ATP phosphorylation assay ab83355: No-wash assay used with cell lysates. Not as sensitive as luminescence assays. Fluorometric (Ex/Em 535/587 nm) is more sensitive than colorimetric. Plate reader.
Oxygen consumption and glycolysis assays
The rate of oxygen consumption indicates the level of cellular metabolic activity. Analysis of intracellular oxygen levels and glycolysis activity allow deeper investigation.
Extracellular oxygen consumption assay ab197243: No-wash assay. Dye signal (Ex/Em 380/650 nm) increases as respiration lowers O2 levels. No need for specialized instruments. Plate reader.
Intracellular oxygen levels assay ab197245: Dye fluorescence (Ex/Em 340/642) is quenched by intracellular oxygen. No-wash assay. Plate reader.
Glycolysis activity assay ab197244: No-wash assay. Lactate production causes extracellular acidification and increased dye fluorescence (Ex/Em 340-380/615 nm). Plate reader.
Learn more with our:
Cytotoxicity assay guide
Test for cell membrane damage, either by measuring the leakage of cellular enzymes or staining with membrane-impermeable dyes.
Cell proliferation and cell cycle assay guide
Monitor the growth of a cell population using cell staining dyes, detect generations of daughter cells, or analyze the cell cycle state of a cell population.
Apoptosis assay / cell death analysis guide
Measure the markers present in different types of cell death.