T 2252

The SGBS pre-adipocyte cell strain was obtained from the laboratory of Prof. Martin Wabitsch et al.^{76 (link)}. SGBS pre-adipocytes were grown to near confluence and then incubated (d0) in a serum-free differentiation medium [2 μmol/l rosiglitazone (Cayman #714740), 25 nmol/l dexamethasone (Sigma Aldrich #D-1756), 0.5 mmol/l methylisobuthylxantine (Sigma Aldrich #I-5879), 0.1 μmol/l cortisol (Sigma Aldrich #H-0888), 0.01 mg/ml transferrin (Sigma Aldrich #T-2252), 0.2 nmol/l triiodothyronine (Sigma Aldrich #T-6397), and 20 nmol/l human insulin (Sigma Aldrich #19278)]. After 4 days the medium was changed, and the cells were further cultured in medium supplemented with 0.1 μmol/l cortisol, 0.01 mg/ml transferrin, 0.2 nmol/l triiodothyronine, and 20 nmol/l human insulin. The SGBS pre-adipocytes were exposed to DEHP [50 µg/ml] (Sigma Aldrich) from d0-d4. DEHP had been solved in DMSO (Sigma Aldrich) in a 1000-fold stock solution. The maximum concentration of DMSO in the culture media was 0.1%. Controls were run as vehicle controls with 0.1% DMSO in the media. The dose used is comparable to DEHP doses found in neonates undergoing clinical procedures, such as transfusion or extracorporeal membrane oxygenation as well as in whole blood and blood components^{77 –79 (link)}. All experiments have been finalized at d8 of differentiation.

PBS (−) was dissolved in 0.8% (w/v) NaCl (Fujifilm Wako Chemicals), 0.02% (w/v) KCl (Nacalai Tesque), 0.28% (w/v) NaHPO₄·12H₂O (Nacalai Tesque), 0.02% (w/v) KH₂PO₄ (Nacalai Tesque), and ultrapure water. PBS (+) was dissolved in 0.1% (w/v) CaCl₂ (Hayashi Pure Chemical Ind., Ltd), 0.1% (w/v) MgCl₂·6H₂O (Katayama Chemical Industries Co, Ltd), 1% (w/v) glucose (Nacalai Tesque), and ultrapure water. PBS was dissolved in 9% (v/v) PBS (−), 10% (v/v) PBS (+), 1% (v/v) PVP (Sigma‐Aldrich), 0.2% (v/v) penicillin streptomycin (Nacalai Tesque), and ultrapure water. Modified NCSU37 (mNCSU37) medium was used as basic medium for pre‐IVM and IVM of porcine COCs.⁵, ²³, ²⁴ Highly purified FSH was a kind gift from the National Hormone and Pituitary Program (National Institute of Diabetes, Digestive and Kidney Diseases; NIDDK). Hypoxanthine (Sigma‐Aldrich) was dissolved in the mNCSU37. Holo‐TF (monoferric or diferric TF; T8158) and Apo‐TF (non‐iron bond TF; T2252) were purchased for Sigma‐Aldrich and were dissolved in the mNCSU37. Fetal calf serum (FCS) was purchased from Invitrogen.