Pcs 210 010

Murine 3T3-L1 and human PCS-210-010 pre-adipocytes obtained from ATCC (Manassas, VA, USA) were, respectively, cultured in DMEM and FBM containing 10% FBS, 2 mmol/L l-glutamine, and 100 units/mL penicillin/streptomycin in humidified incubator at 37 °C with 5% CO₂ and grown to 70−80% confluence before use. Cells between 5 and 17 passages were used in this study.
For adipocyte differentiation, pre-adipocytes were seeded into a 24-well plate at a density of 2 × 10⁴ cells/well. After incubation for 48 h, the differentiation process was counted as day 0, and the cells were treated with differentiation medium containing 0.5 mM IBMX, 1 µM dexamethasone, and 10 µg/mL insulin with or without pinostrobin at non-toxic concentrations. On day 2 of differentiation, the medium was replaced with culture medium containing 10 µg/mL of insulin. On day 4, the cells were further incubated in culture medium with renewal every two days until lipid droplets were obvious upon microscopic examination, approximately on day 8 [35 (link)].

BM cells were flushed out from human fetal femoral bones (Advanced Bioscience Resources), and cultured in DMEM to generate MSCs [46 (link)]. Immunophenotyping of MSCs was analyzed by flow cytometry. To generate mature adipocytes, MSCs were cultured in adipocyte medium (Lonza) for 2 weeks. Adipocytes were also generated from the adipocyte precursor (pre-adipocyte) cell line PCS-210-010 (ATCC), or pre-WAT cells isolated from subcutaneous fat (LONZA), or isolated from the BM aspirates of healthy adults or MM patients, and further purified by 10 min centrifugation at 186 g [47 (link)]. Mature adipocytes were fixed with 4.0% paraformaldehyde, stained with Oil red O for 1 hour, and observed under light microscopy [48 (link)].