Cell Viability Calculator (Trypan Blue and MTT)

Background.

No assay measures "alive" directly. Every viability figure rests on a proxy, and the two proxies used most at the bench answer different questions. Dye exclusion with trypan blue asks whether a cell's outer membrane is intact: healthy cells keep the dye out and look clear under the microscope, while cells with damaged membranes take it up and stain blue. Absorbance assays such as MTT, XTT and neutral red uptake ask whether cells are metabolically active: living cells reduce a yellow tetrazolium salt to a coloured formazan, or concentrate neutral red in their lysosomes, and a plate reader compares the colour with untreated wells.

Different proxies have different blind spots. A cell that has started apoptosis can still exclude trypan blue, and cells that have burst vanish from the count altogether, which flatters a badly damaged sample. A compound that slows metabolism without killing anything lowers an MTT reading; one that reduces the tetrazolium chemically raises it. That is why the calculator keeps the two methods in separate modes and labels each result with what was actually measured.

For absorbance results it uses the equation in ISO 10993-5:2009, the international standard for in vitro cytotoxicity testing of medical devices: viability is 100 times the mean optical density of the treated wells divided by that of the untreated control. The same standard supplies the reference point most people are looking for. Clause 8.5.1 counts a reduction in viability of more than 30 percent as a cytotoxic effect, so a result strictly below 70 percent indicates cytotoxic potential and exactly 70 percent does not. That line was drawn for extracts of medical devices tested under the standard's protocol; a routine culture passaged at 85 percent viability is not "nearly cytotoxic".

What is cell viability calculator?

Cell viability is the percentage of a cell population judged alive by a stated test. In a dye-exclusion count it is the unstained cells as a share of all cells counted, so it can only run from 0 to 100 percent. In an absorbance assay it is the treated wells' signal as a share of the untreated control's, a relative measure of metabolic activity that can exceed 100 percent if the treatment stimulates the cells. The two numbers are not interchangeable, and a viability figure is only reproducible when it is reported with the assay, cell line, exposure time and read wavelength behind it.

How to use this calculator.

1. 01Choose the assay you actually ran. A trypan blue percentage and an MTT percentage measure different things and cannot be converted into each other.
2. 02For dye exclusion, mix the suspension with 0.4% trypan blue, load a counting chamber and count within a few minutes, because longer exposure starts to stain cells that were alive. Enter the clear and blue counts, aiming for at least 200 cells in total.
3. 03To get viable and dead cells per mL, enter the total density of the original suspension, corrected for the dilution made by adding the dye; use the hemocytometer calculator if you counted on a chamber. Enter 0 if you want only the percentage.
4. 04For absorbance, enter the mean optical density of the treated wells and of the untreated control wells, read at the same wavelength: 570 nm with a 650 nm reference for MTT, 450 nm for XTT.
5. 05Leave the background at 0 to follow ISO 10993-5 Equation (C.1) exactly. Enter a cell-free medium-plus-reagent reading only if your procedure subtracts one, and say so when you report the result.

The formula.

Dye exclusion divides a count by a count: V% = L ÷ (L + D) × 100, where L is the unstained and D the stained cells. If you supply a total density, the viable density is that total × V% ÷ 100 and the dead density is the remainder, so the two always add back to the total. The absorbance mode divides a signal by a signal: V% = 100 × (A_t − A_b) ÷ (A_c − A_b), with A_t the treated mean, A_c the untreated control mean and A_b an optional background. With A_b = 0 this is exactly the standard's Viab.% = 100 × OD570e ÷ OD570b. Subtracting the same background from both readings is not neutral: when treated wells read lower than controls, it pulls the ratio further from 100 percent. The ISO margin is V% − 70, in percentage points, and the comparison uses the unrounded value. The calculator refuses a count of zero cells (an empty chamber means the density is below detection, not that every cell died), a control reading at or below the background, and a treated reading below the background, since none of these yields a meaningful ratio. It does not check ISO's acceptance criterion that the control wells' mean OD570 reach at least 0.2; that check is yours.

A worked example.

Frequently asked questions.

References& sources.

1. [1]ISO 10993-5:2009(E), Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity, third edition. Clause 8.5.1 ('Reduction of cell viability by more than 30 % is considered a cytotoxic effect'); Annex A (3T3 NRU protocol; '≥ 70 % of the control group … shall be considered non-cytotoxic'); Annex C clause C.2.5 Equation (C.1), Viab.% = 100 × OD570e / OD570b, for MTT at 570 nm; Annex D clause D.2.5 Equation (D.1) for XTT at 450 nm; clause C.2.3.3 (blank acceptance criterion, mean OD570 ≥ 0.2). Standard text consulted directly 2026-07-29. PAYWALLED for purchase — cited by clause so every figure above is verifiable against the document.
2. [2]Strober, W. (2015). 'Trypan Blue Exclusion Test of Cell Viability.' Current Protocols in Immunology, 111, A3.B.1–A3.B.3. DOI 10.1002/0471142735.ima03bs111, PMID 26529666. The peer-reviewed reference protocol for dye exclusion: cells are suspended in PBS containing trypan blue and scored as clear (viable) or blue (non-viable) cytoplasm, giving the percentage this calculator's dye mode computes. Publisher paywall on the full text; the PubMed record is open.
3. [3]World Health Organization, WHO Laboratory Manual for the Examination and Processing of Human Semen. §2.6 covers vitality assessment by dye exclusion (eosin–nigrosin) and its membrane-integrity basis; §2.8.3 gives the ≥200-cells-per-replicate counting target and the two-significant-figure reporting rule used throughout this page. Public PDF, retrieved 2026-07-29.
4. [4]ISO 20391-1:2018, Biotechnology — Cell counting — Part 1: General guidance on cell counting methods. Establishes the distinction between total and differential counting — which is precisely the distinction between a total cell density and the viable fraction of it that this page computes. PAYWALLED — cited bibliographically; scope confirmed from the ISO catalogue entry, retrieved 2026-07-29.