Effect of ferric citrate and sodium citrate on HSFC proliferation.

Approximately 2 × 10³ HSFCs were added to flat-bottom 96-well plates. The cells were maintained in DMEM with 10% fetal bovine serum, penicillin G, and streptomycin in 5% CO₂humidified atmosphere at 37°C. The cultures were incubated for 9 days in the presence of iron or sodium salt. Cell number was determined on days 1, 3, 5, 7, and 9 using a CellTiter 96 Aqueous nonradioactive cell proliferation assay kit according to the manufacturer's instructions. Briefly, 20 μL combined MTS/PMS solution was added into each well and incubated for 3 hours at 37°C in humidified 5% CO₂atmosphere. Absorbance was measured at 450 nm using an ELISA plate reader.