Isolation and Characterization of Testicular Germ Cells

Testicular single cell suspensions from each sample were obtained by chemical digestion with DNAse I (Serva, Israel), 0.25% EDTA–trypsin solution (1:9, v/v) and mechanical disruption through pipetting through 40-μm cell strainers (Corning, USA) [30 (link)]. The cells were fixed by 3% PFA and permeabilized by 0.2% Tween 20 in PBS. Then, they were labeled with rabbit-anti-mouse VASA (total GC marker) and PLZF (SSPC marker) and c-Kit (differentiating spermatogonia) antibodies for 30 min at RT. For labeling of VASA, the cells were incubated with a secondary FITC-conjugated goat anti-rabbit IgG antibody (Additional file 1: Table 1) at RT for 30 min. Measurements were taken by Novocyte FCM, and data were analyzed with Novoexpress 1.3.0. software, with 10.000 events recorded for each sample. Immune-positive cells were detected by gating according to unstained and isotype control samples.