Viability Assay Calculators

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A viability assay determines the proportion of living cells in a sample or measures cytotoxicity of a drug, treatment, or environmental condition. Cell viability is a critical quality metric in cell culture, drug development, cell therapy manufacturing, and toxicology. Viability can be assessed by membrane integrity (trypan blue exclusion, propidium iodide), metabolic activity (MTT/MTS/WST-1, resazurin, CellTiter-Glo), or apoptosis markers (annexin V, caspase activity). Each method has specific advantages depending on the application, throughput requirements, and available equipment.

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Trypan Blue Exclusion

Live cells with intact membranes exclude trypan blue dye; dead cells take up the dye and appear blue under microscopy. Mix cells 1:1 with 0.4% trypan blue, count in hemocytometer or automated counter (Vi-CELL, Countess). Viability % = live cells / (live + dead) × 100. Fast and inexpensive but labor-intensive at scale and subjective without automation.

MTT Assay (Colorimetric Metabolic Activity)

MTT (yellow) is reduced by mitochondrial dehydrogenases in live cells to formazan (purple insoluble crystals). Crystals are dissolved in DMSO; absorbance at 570 nm is proportional to metabolic activity. Used for proliferation, cytotoxicity IC₅₀ determination. Advantages: high-throughput (96-well plate); disadvantages: endpoint assay (cells killed), multiple wash steps, interference from colored compounds.

Resazurin (alamarBlue/PrestoBlue)

Resazurin (blue, non-fluorescent) is reduced by cellular metabolism to resorufin (pink/red, fluorescent). Can be read fluorescently or colorimetrically; non-toxic at low concentrations, allowing longitudinal measurements of the same culture. Preferred when kinetic data is needed.

CellTiter-Glo (ATP Bioluminescence)

Measures intracellular ATP using luciferase reaction. Luminescence is directly proportional to live cell number. Most sensitive method; works in ≤100 cells; lysis-based (single time point). Used for low-cell-number assays and compound screening in 384/1536-well formats.

Glossary

Cell Viability
The proportion of living cells in a sample: (live cells / total cells) × 100%; measured by membrane integrity, metabolic activity, or ATP content assays.
MTT Assay
A colorimetric metabolic viability assay where yellow MTT is reduced by live cells to purple formazan; absorbance at 570 nm is proportional to metabolic activity; widely used for cytotoxicity IC₅₀ determination.
IC₅₀
The concentration of a compound that inhibits 50% of cell growth or viability compared to untreated control; the standard potency metric in cytotoxicity testing; determined from dose-response curves.

Frequently Asked Questions

A viability assay measures what percentage of cells in a sample are alive, or quantifies how many cells survive a treatment. It is essential in cell culture quality control, drug cytotoxicity testing, CAR-T and other cell therapy manufacturing (where minimum viability is required for release), and toxicology. Common methods: trypan blue exclusion (membrane integrity), MTT/resazurin (metabolic activity), CellTiter-Glo (ATP content), annexin V/PI flow cytometry (apoptosis vs. necrosis discrimination).

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a yellow compound reduced by active mitochondrial dehydrogenases in metabolically active (live) cells to a purple formazan precipitate. After a 2–4 hour incubation, DMSO is added to dissolve the crystals and absorbance is read at 570 nm. Higher absorbance = more metabolically active cells. MTT is widely used for IC₅₀ determination of cytotoxic drugs and is adaptable to 96-well formats, but it is an endpoint assay that kills the cells and requires multiple wash steps.

Trypan blue is a negatively charged dye that cannot cross intact cell membranes. Live cells exclude it and appear clear (unstained) under a microscope. Dead cells with compromised membranes take up the dye and appear blue. Mix equal volumes of cell suspension and 0.4% trypan blue, load onto a hemocytometer (or automated counter), and count blue (dead) and clear (live) cells within the counting grid. Viability % = (live / total) × 100. Limitation: only measures membrane integrity, not functional viability; some metabolically compromised cells may still exclude dye.

Viability assays measure the percentage or number of live cells at a single time point — they answer 'how many cells are alive?' Proliferation assays measure growth over time — they answer 'are cells dividing?' A drug could reduce cell number by either killing cells (cytotoxic) or stopping division (cytostatic) — viability alone cannot distinguish these. Proliferation assays include BrdU/EdU incorporation (DNA synthesis), Ki-67 staining (proliferation marker), CFSE dilution (tracks divisions), and population doubling time calculations. Cytotoxicity assays typically combine viability with a growth curve to distinguish these mechanisms.