Fcm analysis

The apoptotic cells were also evaluated using annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) Apoptosis Detection Kit (BD, USA), to examine early and late apoptotic changes as previously established. The samples were washed twice and adjusted to a 5 × 10⁵ cells/mL concentration with phosphate buffer saline (PBS). 200 μL of suspension was added into each labeled tube. 5 μL of annexin V-FITC and 10 μL PI (20 μg/mL) were then added into the labeled tube and incubated for at least 15 min at room temperature in the dark. 200 μL of cold PBS binding buffer was then added into each tube without washing and analyzed using the FCM analysis (BD, USA), as soon as possible (within 30 min). The apoptotic cells were defined as the population that was PI negative (indicating an intact plasma membrane) and Annexin V-FITC positive. This FCM assay was also done in triplicate.

Podocyte apoptosis was detected by FCM analysis (BD) with a double staining of annexin V‐fluorescein isothiocyanate (FITC) and propidium iodide (PI). After centrifugation, about 5–10 million cells were resuspended in 195 µL annexin V‐FITC binding buffer. Following a 15 min incubation with 5 µL annexin V‐FITC at 4°C in the dark, cells were incubated with 5 µL PI staining solution for 5 min at 4°C in the dark. A tube without annexin V‐FITC/PI was used as a negative control. Through FCM analysis, the rates of apoptotic cells were determined. Annexin V‐FITC was green in fluorescence and marked early apoptotic cells. PI was red in fluorescence. Annexin V⁺/PI⁺ indicated late apoptotic or necrotic cells, Annexin V­/PI⁻ indicated viable cells.