2.3. Gonocyte Isolation and in Vitro Culture and Treatments

Neonatal gonocytes were isolated by performing sequential enzymatic tissue dissociation together with mechanical dissociation of the pooled testes from 40 PND3 pups per experiment. This was followed by a step of differential overnight adhesion at 37 °C in medium containing 5% fetal bovine serum (FBS), and cell separation of the non-adherent cells on a 2–4% bovine serum albumin (BSA) gradient in serum-free medium on the next morning [22,23]. Enriched gonocyte preparations at 70–80% purity were obtained by pooling fractions containing high proportions of gonocytes, according to size and appearance, while a gonocyte purity above 95% was used for gene array analysis [18]. Freshly isolated gonocytes were cultured at 20 to 30,000 cells per well in 500 μL of RPMI 1640 containing 2.5% FBS, antibiotics, alone or with the PRDX inhibitors conoidin A, MJ33 and ezatiostat, and/or H₂O₂, at different concentrations, for 2 to 18 h, in 3.5% CO₂, at 37 °C. Cell differentiation was examined by treating the gonocytes with 10⁻⁶ M retinoic acid (RA), in the absence or presence of the PRDX inhibitors.