Cell viability assay guide
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Learn about your options when you need a cell viability assay.
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
- Dyes reduced by cellular enzymes
- Mitochondrial membrane potential dependent dyes
- Cellular esterase cleaved dyes
- ATP and ADP assays
- Glycolytic flux and oxygen consumption assays
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.
Tetrazolium family
Assay | Instrument | Notes | Assay kits |
MTT | Plate reader | Original tetrazolium assay; still very popular. Only tetrazolium assay that needs a wash/solubilization step. | |
MTS | Most popular assay. More heavily used than WST-1. | ||
WST-1 | More sensitive than MTT, XTT or MTS. | ||
Cell Counting Kit-8/CCK-8/ WST-8 | |||
XTT assay |
Resazurin family
Resazurin is equivalent to the active ingredient of ThermoFisher’s alamarBlue®
Assay | Instrument | Notes | Assay kits |
Resazurin | Plate reader, microscope, flow cytometer | Fluorometric (Ex/Em 535–560/560–615) or colorimetric. No-wash assay. Fluorescent readout enables multiplexing with other assays. |
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.
Assay | Instrument | Notes | Assay kits |
TMRE/TMRM | Plate reader, microscope, flow cytometer | Most popular Abcam mitochondrial membrane dye assay. Ex/Em 549/575 nm. Washed out of mitochondria after fixation. | |
JC-1/JC-10 | 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. | ||
Mitotracker Red | Ex/Em 579 /599. Not washed out after fixation. | ||
Rhodamine 123 | Ex/Em 507/529. Washed out after fixation. | ||
MitoNIR | Plate reader, flow cytometer | Ex/Em 635/660. | |
MitoOrange | Ex/Em 540/590. |
Esterase cleavage
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.
Assay | Instrument | Notes | Assay kits |
Calcein AM | Plate reader, microscope, flow cytometer | Ex/Em 495/515 nm | |
Calcein violet AM | Plate reader, microscope, flow cytometer | Ex/Em 405/460 nm | |
Esterase-cleaved blue | Plate reader | Ex/Em 360/450 nm | |
Esterase-cleaved green | Plate reader, microscope | Ex/Em 490/520 nm | |
Esterase-cleaved near IR | Plate reader | Ex/Em 633/660 nm |
ATP assays
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).
Assay | Instrument | Notes | Assay kits |
Luminescence ATP assay | Luminometric plate reader | No-wash assay. | |
Luminescence ADP/ATP assay | No-wash assay. After ATP analysis, ADP is converted to ATP for detection. | ||
ATP phosphorylation assay | Plate reader | No-wash assay used with cell lysates. Not as sensitive as luminescence assays. Fluorometric (Ex/Em 535/587 nm) is more sensitive than colorimetric. |
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.
Assay | Instru | Notes | Assay kits |
Extracellular oxygen consumption | Plate reader | No-wash assay. Dye signal (Ex/Em 380/650 nm) increases as respiration lowers O2 levels. No need for specialized instruments. | |
Intracellular oxygen levels | Dye fluorescence (Ex/Em 340/642) is quenched by intracellular oxygen. No-wash assay. | ||
Glycolysis activity | No-wash assay. Lactate production causes extracellular acidification and increased dye fluorescence (Ex/Em 340-380/615 nm). |
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.