📋 Workflow
Full workflow, products add-to-cart directly1
Seeding
30 minCount log-phase cells, seed 96-well plate at proper density, add PBS to edge wells to prevent evaporation
💡 Tip Density so control not confluent at endpoint
⚠️ Caution Verify uniformity under microscope
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2
Drug treatment
24–72 hAdd compound in gradient concentrations with negative/positive controls, incubate
💡 Tip 3-6 replicates per concentration
⚠️ Caution Keep DMSO consistent and ≤0.5%
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3
MTT incubation
2 hRemove medium, add MTT solution, incubate 2-4 h at 37°C in dark until formazan forms
💡 Tip Protect from light to prevent MTT degradation
⚠️ Caution Over-incubation makes crystals hard to dissolve
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4
Dissolve and read
30 minRemove supernatant, add DMSO to dissolve crystals, read absorbance at 570 nm
💡 Tip Mix gently to avoid bubbles
⚠️ Caution Use 630 nm reference to subtract background
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⚠️ 5 Common Beginner Mistakes
- ① Attached cells drifting cause uneven OD
- ② Edge well evaporation causes false high viability
- ③ Light-exposed MTT degrades and lowers sensitivity
- ④ High DMSO itself is toxic to cells
- ⑤ Missing reference wavelength inflates readings
❓ FAQ
+MTT vs CCK-8?
MTT needs dissolving, cytotoxic, multi-step; CCK-8 water-soluble, more sensitive, continuous monitoring.
+How to set cell density?
Ensure logarithmic growth without confluence during assay, typically 3k-10k/well.
+OD out of range?
Reduce seeding or incubation so readings fall in linear range (~0.2-1.2).