2.2. Mesangial Cell Culture and Viability

Human renal mesangial cells (HRMC, Sciencell Research Laboratories, Carlsbad, CA, USA) were cultured at 37 °C humidified atmosphere of 5% CO₂ in air. Routine culture of HRMC was performed in DMEM/F12 (7:1) media containing 15% FBS, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. HRMC in passage of 6–10 was sub-cultured at 80% confluence and used for further experiments.

To mimic diabetic glomerular fibrosis caused by chronic hyperglycemia, HRMC was incubated in 33 mM glucose-added DMEM containing 2% FBS and 8 μg g/mL insulin for 3 days in the absence and presence of chrysin. For osmotic control incubations, another set of HRMC was cultured in DMEM containing 2% FBS (+2 μg/mL insulin) and supplemented with 27.5 mM mannitol. In addition, HRMC was incubated in 0.1% BSA media containing 5–100 μg/mL AGE-BSA for 3 days. Culture media was collected and stored at −20 °C. In another set of experiments, HRMC was incubated with 5–100 μg/mL AGE-BSA up to 3 days for diabetic glomerulosclerosis.

After the 3-day incubation period under the condition of 33 mM glucose or 5–100 μg/mL AGE-BSA, the MTT assay was carried out for measuring cell proliferation. After unconverted MTT was removed, the purple formazan product was dissolved in 0.5 mL isopropanol with gentle shaking. Absorbance of formazan dye was measured at k = 570 nm with background subtraction using λ = 690 nm.