Htrf based assay kit

Fed mice were culled, and blood was collected into EDTA-coated tubes (Sarstedt, Beaumont Leys, UK). Glucose measurements were determined using an automatic glucometer (Accu-Chek). For insulin measurements, plasma was separated by centrifugation at 13,200 rpm for 20 min. 5 μl of blood plasma was used to measure insulin levels using an ultrasensitive mouse insulin ELISA kit (CrystalChem). For total islet insulin measurements, five islets were lysed by sonication in acidified ethanol (1.5% HCl (v/v), 75% ethanol (v/v), and 0. 1% Triton X-100 (v/v)). Insulin concentration was determined using a HTRF-based assay kit (CisBio).

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.