MTT / CCK-8 cell viability
Metabolically active cells convert a reagent into a coloured product; its absorbance compares viability. The key is choosing cell number and incubation so the control is neither too faint nor saturated.
Key points
- Keep control OD inside the linear range (about 0.3–2.0)
- Fill edge wells with PBS
- Always include a vehicle (solvent-only) control
Materials
- Clear 96-well plate
- Cells and medium
- Drug stock (e.g. in DMSO)
- MTT (5 mg/mL in PBS) + DMSO, or CCK-8 (WST-8) reagent
- Plate reader (MTT 570 nm, CCK-8 450 nm)
Steps
- 💡 Mix the suspension often while seeding so every well gets the same number.
- 💡 Use the reagent calculator's "Drug stock" and "Serial dilution" tabs for volumes.
🔬 Variable explorer — what changes if…?
The skeleton stays the same, but concentrations, times and reagents change between experiments. Click an item to see what it affects and compare two conditions (A and B) side by side.
Cell number · incubation time
What it isAbsorbance rises with cell number and time until it saturates.
LessToo few or short: low control OD hides drug effects.
MoreToo many or long: the control saturates and toxicity looks weaker than it is.
Typical choiceControl OD about 0.3–2.0. Titrate cell number first for a new line.
Assay type
What it isHow metabolism is measured.
Typical choiceCCK-8 for routine work; ATP luminescence when cells are very few.
Edge effect
What it isOuter wells evaporate faster and drift.
Using edge wellsUsing edge wells for samples: outer wells differ even under identical conditions.
Typical choiceFill the outer ring with PBS/medium and keep the incubator humid.
Solvent (DMSO) concentration
What it isFraction of drug solvent in each well.
HigherHigh: the solvent alone lowers viability and exaggerates drug effect.
Typical choiceSame DMSO % in every well, usually ≤0.1–0.5%.
Dose range
What it isThe range must bracket the IC50 for a stable curve fit.
Too narrowToo narrow: no top/bottom plateaus and an unstable IC50.
Typical choice8–10 steps at 3–10-fold spacing, from no effect to full inhibition.
Troubleshooting
| Problem | Common cause | Fix |
|---|---|---|
| Control OD too low | Too few cells, short incubation | More cells or longer incubation |
| Saturated OD (> 2.5) | Too many cells, long incubation | Fewer cells, shorter incubation |
| Large replicate spread | Uneven seeding, bubbles, edge wells | Mix while seeding, remove bubbles, skip edges |
| Viability above 100% at low doses | Wrong reference, mild growth stimulation | Use vehicle as 100%; do not force the top to 100 |
⚠ Safety MTT and drugs may be toxic, and DMSO carries substances through skin — wear gloves.
This page summarises widely used general conditions. Reagent, kit and antibody manuals and your institution's safety rules take priority. Simulators marked "concept" are simplified models that show trends.
Analyse the results with free tools:
IC50 calculator → Drug stock & DMSO % → Serial dilution →