Flow Cytometry

Cells that were harvested from the total testicular tissues and culture were considered. After trypsin treatment, cell number was calculated and a minimum of 1 × 10⁵ cells were considered per tube (n = 3). Cells in tubes were washed with fluorescence‐activated cell sorting (FACS) buffer (2 times). Cells in one tube were fixed with 2% paraformaldehyde (PFA) at this stage; the other two tubes were fixed with BD Cytofix/Cytoperm (Ct No. 554714, San Jose, CA, USA) for 15 min at RT. After washing them two times with perm wash, primary antibodies against PDGFRA, 3BHSD, SOX9, and αSMA were added and cells were incubated for 30 min. Again, cells were washed with perm wash and blocked with Fc receptor block for 20 min, after which secondary antibodies were added and cells were incubated for 30 min. After incubation, cells were washed with FACS buffer (three times), fixed with PFA, and suspended in FACS buffer before analyzing using FACS.