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Cell health

Cell viability assay guide

​​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

See below to learn more about these assay methods, or review our most popular cell viability assay kits based on MTSresazurinTMREcalcein 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.

ab211091

MTS

Most popular assay. More heavily used than WST-1.

ab197010

WST-1

More sensitive than MTT, XTT or MTS.

ab155902
ab65473
ab65475

Cell Counting Kit-8/CCK-8/ WST-8


ab228554

XTT assay


ab232856

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.

ab129732


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.

ab113852

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.

JC-1: ab113850
JC-10:
ab112134
ab112133

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.

ab112149
ab112150

MitoOrange

Ex/Em 540/590.

ab138898
ab138899


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

ab228556

Calcein violet AM

Plate reader, microscope, flow cytometer

Ex/Em 405/460 nm

ab176748

Esterase-cleaved blue

Plate reader

Ex/Em 360/450 nm

ab112120

Esterase-cleaved green

Plate reader, microscope

Ex/Em 490/520 nm

ab112122

Esterase-cleaved near IR

Plate reader

Ex/Em 633/660 nm

ab112123


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.

ab113849

Luminescence ADP/ATP assay

No-wash assay. After ATP analysis, ADP is converted to ATP for detection.

ab65313

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.

ab83355


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
ment 

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.

ab197243

Intracellular oxygen levels

Dye fluorescence (Ex/Em 340/642) is quenched by intracellular oxygen. No-wash assay.

ab197245

Glycolysis activity

No-wash assay. Lactate production causes extracellular acidification and increased dye fluorescence (Ex/Em 340-380/615 nm).

ab197244


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.


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