A resazurin-based cell viability assay of mitochondrial DNA-depleted glioma C6ρ0 cells

Abstract

Objective To establish a method for determining the cell viability of mitochondrial DNA (mtDNA)-depleted C6 cells (C6ρ0) using resazurin as the indicator of redox. Methods We performed a systematic analysis to determine whether C6ρ0 cells were able to reduce resazurin and whether there was a linear correlation between resazurin reduction and cell viability. The conditions of the resazurin assay were optimized by evaluating the fluorescence intensities in cell cultures with different cell numbers, varying concentrations of resazurin, and after different incubation time. This resazurin assay was assessed for its sensitivity in detecting the viability of C6ρ0 and C6 cells in comparison with MTT assay. Results In resazurin assay, an optimal linear relationship between the cell number and the fluorescent intensity was maintained when the cell density was 0~30 000 per well and the cells were incubated in a 96-well plate with 25 μg/mL resazurin in the dark for 1 to 4 h at 37 ℃ with 5% CO2. In the cytotoxicity test of ursolic acid using resazurin assay, ursolic acid significantly inhibited the proliferation of C6ρ0 and C6 glioma cells (P < 0.05) with IC50 values of 20.82 μg/mL and 14.62 μg/mL, respectively. Conclusion The resazurin assay allows sensitive, nontoxic and cost-effective determination of the cell number of mtDNA-depleted cells, and can also be used for cell proliferation and viability assessment, with high-throughput screening of cytotoxic compounds.

Keywords

• resazurin
• c6 glioma
• c6ρ0
• cell viability
• ursolic acid