Medium 199

Eight-week-old BALB/c female mice were provided by Boyuan Experimental Products Department (Hefei, China). The mice were weighed and executed by cervical dislocation to obtain the ovaries. All animal experiments were approved by the Institutional Animal Care and Use Committee of Anhui Medical University. After washing the tissues with phosphate-buffered saline (PBS, Gibco, USA) twice at room temperature, ovary cryopreservation was performed using Medium 199 (Sigma, USA) supplemented with 100 IU/ml penicillin and streptomycin (Hyclone, USA) for 10 min [21 (link)]. And ovaries were transferred to 10 ml of 5% dimethyl sulfoxide (DMSO, Sigma, USA) and 5% ethylene glycol (EG, Sigma, USA) and 10 ml of 10% DMSO and 10% EG in Medium 199 for 10 min respectively [22 (link), 23 (link)]. Each ovary was directly put into an open vessel filled with liquid nitrogen, and the vitrified ovarian tissue was put into the precooled marked freezing tube. After tightening the lid, the freezing tube was put into liquid nitrogen for storage. Ovaries were thawed 7–10 days later. Freezing tubes were first immersed in a 37 °C water bath with gentle shaking. And ovaries were transferred in turn to the following three thawing solutions for 5 min respectively: (i) 10 ml of 0.5 M sucrose in Medium 199, (ii) 10 ml of 0.25 M sucrose in Medium 199, and (iii) 10 ml Medium 199 [24 (link), 25 (link)].

Primary hepatocytes were isolated from 8-week-old male C57BL/6N mice by collagenase perfusion [7 (link)]. The cells were maintained in Medium 199 (catalogue no. M4530; Sigma-Aldrich, UK) supplemented with 10% (vol./vol.) FBS, 1% (vol./vol.) antibiotics and 10 nmol/l dexamethasone, and were infected with adenovirus for 24 h. Subsequently, the cells were maintained in Medium 199 without 10% FBS and were treated with 25 mmol/l glucose (catalogue no. G7021; Sigma-Aldrich) and/or 100 nmol/l insulin (catalogue no. I6634; Sigma-Aldrich) for 48 h, 250 μmol/l palmitic acid (catalogue no. P9767; Sigma-Aldrich) for 24 h, 300 μmol/l AICAR (catalogue no. A611700; Toronto Research Chemicals, Canada) for 1 h, or 1 μmol/l T0901317 (catalogue no. T2320; Sigma-Aldrich) for 24 h.

The murine muscle myoblast cell line C2C12 was purchased (ATCC, VA, USA) and maintained in proliferation media, composed of Dulbecco’s Modified Eagle’s Medium (DMEM) 25 mM glucose (Lonza, UK) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo, UK) and 1% Penicillin/Streptomycin (P/S) (Thermo, UK). Upon cells reaching 70–80% confluence, the differentiation medium, composed of DMEM 25 mM glucose supplemented with 2% horse serum (HS) (Thermo, UK) and 1% P/S, was added to induce differentiation. The myoblasts were allowed to differentiate for 6 days with fresh differentiation medium added every other day, sufficient for the successful development of mature myotubes. The human muscle myoblast cell line LHCN-M2 (Evercyte, Germany) was grown in proliferation media as described [58 (link)], composed of a 4:1 ratio of DMEM and Medium 199 (Sigma-Aldrich, UK) supplemented with 15% FBS, 200 mM HEPES (Thermo, UK), 0.03 µg/ml zinc sulfate (Sigma-Aldrich, UK), 1.4 µg/ml vitamin B12 (Sigma-Aldrich, UK), 0.055 µg/ml dexamethasone (Sigma-Aldrich, UK), 2.5 ng/ml hepatocyte growth factor (Proteintech, UK), 10 ng/ml basic fibroblast growth factor (Sigma-Aldrich, UK) and 1% P/S. The differentiation media of LHCN-M2 was composed of a 4:1 ratio of DMEM and Medium 199 supplemented with 2% HS and 1% P/S. LHCN-M2 differentiation was performed similarly to C2C12 myoblasts.