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For robust results, we recommend our optimized MTT assay kit ab211091:
An expanded segment of the assay data at lower cell number per well:
HeLa cells were grown in DMEM media supplements with 10% FBS, harvested using trypsin, and counted using Trypan blue and a hemocytometer. Cells were then serially diluted in a clear cell culture plate and incubated for 3 hours with MTT reagent at 37°C. After incubation, cells were treated with MTT solvent for 15 minutes at room temperature. Absorbance was measured at OD = 590 nm.
Alternatively, we recommend the protocol below.
Prepare MTT solution
MTT is soluble in water (10 mg/mL), ethanol (20 mg/mL), and buffered salt solutions and culture media (5 mg/mL). We recommend using a 5 mg/mL solution in PBS. Mix by vortexing or sonication.
Filter sterilize solution after adding MTT.
Store MTT solution at -20°C (stable for at least 6 months). Do not store at 4°C for more than a few days.
Prepare MTT solvent
4 mM HCl, 0.1% NP40 in isopropanol
Cell proliferation assays
For cell counting, a standard curve can be established with known cell number and fixed incubation times with the assay reagent.
Cell cytotoxicity assays