Annexin V and PI Staining and Flow Cytometry

The percentages of early and late apoptotic cells in human SSCs transfected without or with treatment of miR-663a mimics, miR-663a inhibitor, miRNA mimics control, miRNA inhibitor control, NFIX siRNAs, or control siRNA were measured using the APC Annexin V and PI apoptosis detection kit and flow cytometry, according to the manufacturer’s instruction. Human SSC cells were seeded at a density of 5 × 10⁴ cells/well in 12-well plates. Cells were collected by centrifuging at 1,000 rpm for 5 min and washed twice with PBS at day 3 after transfection. The cells were simultaneously stained with Annexin V-FITC (green fluorescence) and the non-vital dye PI (red fluorescence), which allowed the identification of intact cells (FITC⁻PI⁻), early apoptotic cells (FITC⁺PI⁻), and late apoptotic cells (FITC⁺PI⁺).