Before you start

Seed cells at a density that stays in log growth for the whole assay (often 5,000–10,000 cells/well).
Include untreated, vehicle and blank wells.

Reagents and materials

Totals are for 96 wells (+10% extra). Per-well amounts are in the middle column.

Reagent	Per well	Total
MTT, 5 mg/mL in PBS (filtered, protected from light)into 100 µL medium (0.5 mg/mL final)	10 µL	10 µL
DMSO (solubilisation)	100 µL	100 µL

Viability (%)	(A570 sample − A570 blank) ÷ (A570 untreated − A570 blank) × 100
Blank	medium + MTT, no cells

Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.

Treat cells
Treat for the planned time.
Add MTT
Add 10 µL MTT stock per 100 µL medium.
Incubate
Incubate 2–4 h at 37 °C until purple formazan crystals form.
Solubilise
Remove medium carefully (adherent cells) and add 100 µL DMSO; for suspension cells use an SDS/HCl solubilisation buffer instead.
Mix
Shake 10–15 min protected from light until crystals dissolve.
Read
Read absorbance at 570 nm with a 630–690 nm reference.

MTT reflects metabolism, not cell number directly — metabolic drugs can bias it.
Keep cells out of over-confluence.

Problem	What to check or change
High variability	Seed evenly, avoid edge wells or fill them with PBS, dissolve crystals fully.
Low signal	Seed more cells or extend MTT incubation.

MTT is harmful; DMSO penetrates skin — wear gloves.