Isolation of Testicular Single Cells

Testes were collected in ice-cold PBS and the tunica albuginea was removed carefully. Testicular tissue was minced in 200 μl of digestion medium [1 mg/ml collagenase/dispase (10269638001, Roche), 1 mg/ml hyaluronidase (H3506; Sigma-Aldrich), 1 mg/ml DNAse I (DN-25; Sigma-Aldrich) in DMEM] until all seminiferous tubules seemed to be digested. Then 800 μl of digestion medium was added and incubated for 25 min at 37°C with slow continuous rotation. Additional mechanical disruption was performed by pipetting the solution five times using recovery tips. The suspension was filtered through a 35 mm pore size filter to achieve a single-cell suspension. The cell suspension was pipetted slowly to avoid clogging and cell damage. Cells were collected by centrifugation at 400 g for 10 min at 37°C, the supernatant was discarded, and the pellet was resuspended in 1 ml of PBS (Umer et al., 2021 (link)).