Isolation and Analysis of Testicular Interstitial Cells

Testicular interstitial cells were prepared as previously described [23] (link), [24] (link). Briefly, testes were treated with type I collagenase (1 mg/ml, Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 37°C for 30 min and resultant single suspension cells were separated with a 40%/70% discontinuous Percoll density gradient (GE Healthcare, Uppsala, Sweden). These cells were first stimulated with phorbol 12-myristate 13-acetate and ionocymin for 3 h in the presence of brefeldin A, stained with antibodies against cell surface antigens, fixed, and permeabilized using the staining buffer set according to the manufacturer’s instructions (eBioscience). Antibodies against cell surface antigens of CD4, CD8, CD45, B220 and F4/80 (Biolegend, San Diego, CA, USA, or eBioscience) were used after blocking nonspecific Fc binding with anti-CD16/CD32 (2.4G2, American Type Culture Collection, Manassas, VA, USA). These cells were then intracellularly stained with antibodies against IFN-γ, IL-17, and Foxp3 (Biolegend or eBioscience) and analyzed with the FACSCanto II flow cytometer (BD Biosciences). Data analysis was performed with the FlowJo 9.6.2 software (Tree Star, Ashland, OR), and the absolute number of positive cells per testis was calculated from percentages obtained by flow cytometric analysis and total number of interstitial cells.