Cell viability calculator
Percent viability from trypan blue counts, MTT absorbance or LDH release.
Percent viability from trypan blue counts, MTT absorbance or LDH release.
Cell viability is the percentage of cells in a sample that are alive. There are two common ways to measure it, and they use different formulas. A dye exclusion count such as trypan blue looks at individual cells and gives the live fraction directly. Aplate assay such as MTT measures a signal produced by living cells and expresses treated wells as a percentage of untreated control wells. This calculator does both, and adds the LDH release assay, which measures dead cells instead of live ones.
Trypan blue cannot cross an intact membrane, so live cells stay clear and dead cells turn blue. Count both in the same large squares of the hemocytometer. Each 1 mm × 1 mm square under a 0.1 mm coverslip gap holds 0.1 µL, which is where the factor 10⁴ comes from. Mixing equal volumes of cell suspension and 0.4 % trypan blue is a dilution factor of 2.
Worked example. 342 live and 38 dead cells in 4 large squares, mixed 1:1 with dye. Viability = 342 / 380 × 100 = 90.0 %. Viable density = (342 ÷ 4) × 10,000 × 2 =1.71 × 10⁶ viable cells/mL; counting the dead cells too, the total is 1.90 × 10⁶ cells/mL. In 5 mL of suspension that is 8.55 million viable cells.
The calculator also gives a 95 % confidence interval for the viability (Wilson score interval), which depends only on how many cells were counted. With 380 cells it is 86.6 to 92.6 %; with 40 cells the same 90 % would span 77 to 96 %. Count at least 100 cells, and count within 3 to 5 minutes of adding the dye because trypan blue is toxic on longer exposure. For other chamber types, several squares with separate counts, or the volume to seed a plate, use the hemocytometer calculator.
In an MTT assay, dehydrogenases in metabolically active cells reduce the yellow tetrazolium salt to purple formazan, read at 570 nm after solubilisation. The absorbance is proportional to the number of living cells within the linear range of the assay. The blank is a well with medium and reagent but no cells; its absorbance is subtracted from every other well before the ratio is taken. The control is untreated (or vehicle-treated) cells and defines 100 %.
Worked example. Control wells 0.85, 0.88 and 0.82 average 0.850, blank wells average 0.050 and treated wells 0.62, 0.60 and 0.64 average 0.620. Viability = (0.620 − 0.050) / (0.850 − 0.050) × 100 = 71.3 %, so the treatment reduced the signal by 28.7 %. The standard deviation, propagated from the replicate wells of both groups, is ±3.7 %.
The same arithmetic applies to MTS, XTT, WST-1, WST-8 (CCK-8), resazurin (alamarBlue, fluorescence) and ATP luminescence (CellTiter-Glo): enter the raw plate reader values. Paste the replicate wells of each group separated by spaces or commas. Add one row per treatment or concentration to get a table; to turn a concentration series into a half-maximal value, take the viabilities to the IC50 calculator.
Lactate dehydrogenase leaks out of cells whose membrane has failed, so the LDH activity in the supernatant measures dead cells. Spontaneous release comes from untreated cells and maximum release from cells lysed with detergent. A sample reading of 0.75 with spontaneous 0.25 and maximum 1.45 gives (0.75 − 0.25) / (1.45 − 0.25) × 100 =41.7 % cytotoxicity. A medium-only background, if subtracted from all three readings, cancels out of this formula.
| Assay | Measures | Good for | Watch out for |
|---|---|---|---|
| Trypan blue | Membrane integrity, cell by cell | Routine passaging, counting before seeding | Subjective near the threshold; misses early apoptosis |
| MTT / MTS / CCK-8 | Metabolic reduction of a tetrazolium dye | Drug screens in 96-well plates | Reports metabolism, not cell number; compound interference |
| Resazurin | Metabolic reduction, fluorescent | Time courses, since cells survive the assay | Over-incubation saturates the signal |
| LDH release | Enzyme leaked from dead cells | Cytotoxicity, membrane damage, short exposures | Serum contains LDH; enzyme decays over days |
For a dye exclusion count, viability (%) = live cells / (live cells + dead cells) × 100. For a plate assay such as MTT, viability (%) = (treated absorbance − blank) / (control absorbance − blank) × 100. The first is the share of cells that are alive; the second is the signal of treated wells relative to untreated wells.
Count unstained (live) and blue (dead) cells in the same hemocytometer squares. Viability is live / (live + dead) × 100. Viable cells per mL is the mean live count per large square × 10,000 × the dilution factor, which is 2 for a 1:1 mix of cells and dye. With 342 live and 38 dead cells over 4 squares, viability is 90 % and the suspension holds 1.71 million viable cells per mL.
Average the replicate wells of each group, subtract the mean blank (medium plus reagent, no cells) from both, and divide treated by control: (0.620 − 0.050) / (0.850 − 0.050) × 100 = 71.3 %. The same formula applies to MTS, XTT, WST-1, CCK-8 and resazurin (alamarBlue) readings.
Treated wells gave more signal than the untreated control. Common causes are a treatment that stimulates proliferation or mitochondrial activity, a compound that reduces MTT chemically or absorbs near 570 nm, and uneven seeding or edge-well evaporation. Run a cell-free well with the compound to check for interference.
A healthy routine culture is normally above 90 percent viable by trypan blue, and most protocols for transfection, flow cytometry or single-cell sequencing ask for at least 80 to 90 percent. Below about 70 percent the culture is in trouble: check for contamination, over-confluence, harsh trypsinisation or a poor freeze-thaw.