MTT assay to detect hepatocyte proliferation

Freshly isolated hepatocytes were adjusted to 1×10⁶/ml cell density in 10% FBS RPMI-1640 medium. Trypan blue staining to detect live hepatocytes showed more than 95% cell viability. The suspended hepatocytes were seeded into 96-well plates by distributing 100 μl into each well. Each experimental group had five parallel samples and the cells were incubated at 37°C and 5% CO₂ under standard cell culture conditions. MTT solution (5 mg/ml) was added into the 96-well plates with 20 μl per well and incubated for 4 h. After the supernatant was discarded, 100 μl dimethyl sulfoxide was added to each well to dissolve blue-purple formazan crystals. The absorbance of each sample was measured by reading the results at 492 nm wavelength using a microplate reader. The absorbance detected can be used to represent the proliferation activity.