Bca protein quantification kit

GCs were cultured in 96-well dishes (1 × 10⁵ viable cells/150 μL media/well) in modified McCoy’s 5A medium in the presence or absence of DHA (1, 10, 20 or 50 μM) or TUG-891 (1, 10 or 50 μM) for 48 h. Supernatants and cells were separately stored at − 20 °C until progesterone analysis and protein assays (BCA protein quantification kit;Interchim, Montluçon, France), respectively. The concentration of progesterone was determined in the culture media using an ELISA protocol, as described previously [44 ]. For progesterone concentrations ranging from 0.25 to 32  ng/mL, the intra-assay coefficient of variation (CV) averaged < 10%. Progesterone secreted in each well was normalized by the protein concentration of the same well. The results are expressed as the amount of progesterone (ng/mL) secreted per 48  h per protein amount (μg/mL) per well. Data, representing 12 independent cultures (as described in GC-proliferation section) with each treatment conducted in quadruplicate, are expressed as mean ± SEM and as ng of secreted progesterone per μg of protein.

Following two washing steps with ice-cold PBS, 2 × 10⁵ cells were scraped off in 200 µL ice-cold PBS containing 2 mM EDTA. Cell suspensions were lysed with 3% sulfosalicylic acid for 10 min on ice and centrifuged for 10 min at 14,000× g and 4 °C. The supernatant was incubated with triethanolamine/H₂O (1:1) and loaded with assay mix (300 µM DTNB, 450 µM NADPH, 20 units glutathione reductase (Sigma)) on a 96-well plate. A standard curve was generated by measuring different concentrations of pure GSH (Sigma). Absorption was measured at 390 nm for 26 min with a Tecan GENios reader. To determine total protein concentration, pellets were solved in 0.2 N NaOH and incubated overnight at 37 °C and subsequently measured using the bicinchoninic acid (BCA) protein quantification kit (Interchim, Montluçon, France) and BSA as standard. Statistical significance was calculated with a paired t-test (two-tailed) using GraphPad Prism software (n = 5; * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001).