Annexin V/PI staining assay

Cellular apoptosis was detected with a flow cytometer using the annexin V/PI staining assay kit. Briefly, HK-2 cells (1.6 × 10⁵) were seeded in 6-well plates and treated with various concentrations of rhein for 24 h. Cells were subsequently collected and re-suspended in 200 μL binding buffer. Then the cells were incubated with 5 μL annexin V-FITC and 10 μL PI (propidium iodide) in the dark for 10 min at room temperature. The amounts of live, apoptotic and necrotic cells were determined by using a flow cytometer (Becton Dickinson, CA, USA) at 525 nm and 575 nm.