Tm3 cells

Mouse Leydig cell TM3 cells were obtained from American Type Culture Collection (Manassas, VA) and routinely maintained in a 1:1 mixture of Ham's F12 medium and DMEM supplemented with 2.5 mM l-glutamine, 15 mM Hepes, and 10% FBS (Thermo Fisher Scientific, Carlsbad, CA). Mouse primary Leydig cells were harvested from the testes of male mice (8–12 weeks old) as previously described (21 (link)). C57BL6/J male mice were maintained under specific pathogen-free conditions in accordance with the international guidelines approved by the Ewha Womans University's Institutional Animal Care and Use Committee (IACUC 17-013). Mouse testes were decapsulated and incubated with collagenase A (0.25 mg/ml) in a shaking water bath at 37°C for 15 min, and mouse Leydig cells were purified by discontinuous Percoll gradients of single-cell suspensions. Cells were resuspended in DMEM/F-12 and filtrated through a nylon mesh (100 μm pore), followed by plating with 2 × 10⁶/ml cell density. Cells were refreshed every day and cultured for 3 days.

The TM3 cells used in the experiments were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured using a 1:1 mixture of Dulbecco’s modified Eagle`s medium (Gibco, Waltham, MA, USA) and F-12K medium, to which 5% fetal bovine serum (Merck Millipore, Berlin, Germany), 5% horse serum (Welgene, Seoul, Korea), and 1% antibiotics (penicillin/streptomycin, Gibco) were added. Cells were cultured in a cell incubator (INS 153, Memert, Schwabach, Germany) at 95% humidity, 5% CO₂, and 37°C.