Testosterone Measurement in Leydig Cells

Testosterone levels were measured using an HTRF-based assay Kit (CisBio Bioassays, Codolet, France) using the same method as in our previous work [30 (link)]. Primary Leydig cells in serum-free RPMI were cultured in 96-well plates at 120,000 cells/well and then incubated for 1 h with or without KH7, 2-CE, 4-CE, LRE1, or ddAdo5′. They were then stimulated with hLH (0.7 nM), or only with serum-free RPMI (control) for 3 h at 35 °C. Afterwards, 10 µL of culture supernatant were transferred to a 384-well white microplate and 5 µL of testosterone-XL665 + 5 µL of anti-testosterone-Ey3 + cryptate antibody were added. The 384-well microplate was then incubated at room temperature in the dark for 1 h, excited with a Mithras LB 943 plate reader (Berthold Technologies GmbH & Co. Wildbad, Germany) at 320 nm, and finally fluorescence was measured at 620 nm and 665 nm.