Viability & cytotoxicity
Living cells reduce a yellow tetrazolium to coloured formazan. MTT gives insoluble purple crystals that must be dissolved (DMSO or acidified SDS) before reading at 570 nm; MTS and similar salts give a soluble product read directly at about 490 nm.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
MTT / MTS
Living cells reduce a yellow tetrazolium to coloured formazan. MTT gives insoluble purple crystals that must be dissolved (DMSO or acidified SDS) before reading at 570 nm; MTS and similar salts give a soluble product read directly at about 490 nm.
How it differs from the standard method
- MTT: remove medium, dissolve crystals, read A570 with a 650 nm reference.
- MTS/XTT/WST: add reagent, incubate 1–4 h, read without a solubilisation step.
- Both report metabolic reducing capacity, which a mitochondrial drug can change without killing anything.
1A well-level proxy for living cells
Plate viability assays do not count cells. Each one measures something that living cells have and dead cells lose, summed over a whole well, and compares it with wells that received only the vehicle.
Reducing activity
Tetrazolium → formazan, read as absorbance (570 nm for MTT after dissolving, 490 nm for the soluble MTS product). Cheap, but it measures metabolism, not cell number.
Luminescence
Add-mix-read: the reagent lyses the cells and luciferase makes light in proportion to ATP. The most sensitive and the widest linear range; needs opaque white plates.
Membrane damage
Enzyme activity in the supernatant, scaled between spontaneous and maximum release. Reports cells that died, not cells that stopped growing.
Single-cell dyes
A membrane-permeant esterase substrate (calcein-AM) marks live cells green, an impermeant DNA dye marks dead ones; counted by imaging or flow.
2From readings to an IC50
Each well's reading has the no-cell blank subtracted and is expressed as a percentage of the vehicle wells, which carry the same solvent as the treated wells. Percent viability against log dose usually gives a sigmoid, fitted with a four-parameter logistic: top plateau, bottom plateau, Hill slope and the midpoint, the relative IC50.
3How the numbers go wrong
- Overgrown vehicle wells: confluent cells stop dividing and reduce less MTT each, so drug-slowed wells catch up and the IC50 moves to higher doses.
- A dose series that stops short of the bottom plateau, leaving the fit to invent one.
- Solvent above about 0.5% DMSO acting as a treatment of its own, hidden inside every normalised value.
- Edge wells evaporating over two or three days, especially when they hold the vehicle reference.
- Compounds that reduce MTT themselves, absorb at the read wavelength or inhibit luciferase: a cell-free well with the top dose catches them.
- Signal outside the reader's linear range: formazan above A ≈ 2, or MTT used up in a crowded well.
Key terms
Practise these as flashcards →Concentration that reduces the response halfway between the fitted top and bottom (relative), or to 50% of vehicle (absolute).
Cells given the solvent alone at the same final concentration as the treated wells; the 100% reference.
Sigmoid with top, bottom, midpoint and Hill slope, fitted to response against log dose.
1 − 3(σ₊ + σ₋)/|μ₊ − μ₋| for positive and negative controls; a measure of the assay window.
LDH in the medium of untreated wells, and in wells lysed with detergent; the 0% and 100% of an LDH assay.
Different evaporation and temperature in the outer wells of a plate, which shifts their readings.
When to use it
- Ranking compounds or doses by potency on a cell line, where a 96- or 384-well IC50 is the deliverable.
- Checking that a treatment used for another experiment (a knockdown, an inhibitor) is not simply killing the cells.
- Separating death from growth arrest: pair a viability readout (ATP or MTT) with a death readout (LDH or a live/dead stain).
Compared with alternatives
Trypan blue counts single cells in a suspension; use it for routine culture, not for dose series across a plate.
When you need to know how the cells die (caspase activation, phosphatidylserine exposure), not just how many are left.
When the compound may be cytostatic: EdU or a DNA-content profile shows whether cells stopped in a phase rather than died.
The instrument side: gain, integration and linear range for the absorbance or luminescence read.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Living cells reduce a yellow tetrazolium to coloured formazan. MTT gives insoluble purple crystals that must be dissolved (DMSO or acidified SDS) before reading at 570 nm; MTS and similar salts give a soluble product read directly at about 490 nm.
- MTT: remove medium, dissolve crystals, read A570 with a 650 nm reference.
- MTS/XTT/WST: add reagent, incubate 1–4 h, read without a solubilisation step.
- Both report metabolic reducing capacity, which a mitochondrial drug can change without killing anything.