Its liquid media supplement

Ovaries were collected from 8dpp CD1 mice, cleared of the surrounding tissues and incubated in 1 mg/mL collagenase type I (Sigma-Aldrich) in αMEM for 1 hr at 37°C in agitation. Following collagenase removal, the ovaries were suspended in the cultured medium (see below) and disaggregated by gently pipetting. Cell suspension was filtered through a 40 μm nylon filter (BD Falcon) while the retained tissue fragments were further incubated in 0.25% trypsin (Sigma-Aldrich) for 15 min at 37°C in agitation, disaggregated and filtered as above. The cellular suspensions were then mixed and finally plated at 30.000 cells/cm². Culture was carried out in αMEM (Aurogene) supplemented with 10% FBS (Gibco), L-glutamine, penicillin-G and streptomycin, pyruvic acid, N-acetyl-L-cysteine (all from Sigma-Aldrich) and ITS liquid media supplement (Gibco), at 37°C in 5% CO₂ in 95% humidified incubator. The day after ( = T0 time point), 0.5 μM EPI, 10 μM CS or 10 μM PM was added to the culture medium of the treated groups. Where indicated, 200 mIU/mL LH was also added to the medium 1 hr before drugs.
Images were acquired under phase contrast microscope (Leitz Diavert) equipped with a DS-Fi1 camera (Nikon).

Human HEK 293T cells, human HepG2 cells, Rat HSC-T6 cells and mouse RAW264.7 macrophage cells were cultured in DMEM/H (Gibco) with 10% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Gibco), and 1% penicillin-streptomycin (P/S, Gibco), in a 5% CO₂ atmosphere at 37 °C. Mouse AML12 were grown in DMEM/F12 with 10% FBS, 1% P/S, and 1% ITS Liquid Media Supplement (Gibco, 51300044) in a 5% CO₂ atmosphere at 37 °C. Polyethylenimine (PolySciences, 23966) was used for transfecting according to the instructions of manufacturer.