Annexin V-FITC/PI Apoptosis Assay

The apoptosis percentages of male germ cells of wild type and P63^(+/−) mice were detected using the Annexin V-FITC/PI kit by flow cytometry pursuant to the manufacturer’s instruction (Biolegend, London, UK). Male germ cells were obtained from P63^(+/−) mice and wild-type mice using two-step enzyme and followed by the differential plating and culture for 24 h. It was feasible to identify and quantify apoptotic cells by conjugating FITC to Annexin V using flow cytometry. The cells were simultaneously stained with Annexin V-FITC (green fluorescence) and the non-vital dye PI (red fluorescence), which allowed the discrimination of intact cells (FITC⁻PI⁻), early apoptotic (FITC⁺PI⁻) and late apoptotic or necrotic cells (FITC⁺PI⁺). The procedure was carried out according to the protocol as described previously^{77 (link)}.