Murine 3t3 l1 cells

Murine 3T3-L1 cells were obtained from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan, ROC). Cells were seeded on the 6-well-plate at 1 × 10⁵ cells/well and cultured in Dulbecco's modified eagle medium (DMEM; Himedia, Mumbai, India) supplemented with 10% heat-inactivated bovine serum (Gibco, ThermoFisher, Grand Island, NY, USA). When reaching full confluence, cells were cultured with the differentiation medium composed of 0.2 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 10 μg/ml insulin in 10% heat-inactivated fetal bovine serum (Gibco, ThermoFisher) DMEM. This differentiation medium was removed after 4 days and replaced with folic acid-free DMEM (Himedia) plus 10 μg/ml insulin either containing 0 μM (f₀), 9.1 μM (f₁), 45.3 μM (f₅), or 90.6 μM (f₁₀) folic acid. The medium was replenished every 2 days until adipocytes had acquired intracellular lipid droplets after 12-day incubation. For the proinflammatory cytokines experiment, preadipocytes were seeded on the 24-well-plate at 1.5×10⁴ cells/well-cultured with f₀ or f₁ medium, and the supernatant was collected for cytokines assay after 4 days. A number of four to five independent experiments were performed for the in vitro folate deficiency study.

Murine 3T3-L1 cells, purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan), were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 1% antibiotics (PEN-STREP-AMPHO), 1 mM sodium pyruvate, 4.5 g/L glucose and 10% fetal bovine serum under 37 °C, 5% CO₂ and 95% humidity until confluent. Differentiation was induced by changing the medium to DMEM supplemented with 0.5 mM 3-isobutyl-1-methylxanthine, 1 mM dexamethasone, 10% fetal bovine serum, and 10 mg/L insulin for the next 2 days, then differentiation medium was replaced with previous maintenance medium and medium was changed every 48 h until about 8 days. The differential process was as described [31 (link)] with modification.