Iscove s modified dulbecco s medium

Primary human epidermal keratinocytes (HEKn; American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured in growth medium (GM) consisting of dermal cell basal medium (ATCC) supplemented with a keratinocyte growth kit (ATCC). Cultures were maintained at 37 °C and 5% CO₂.
Human dermal fibroblasts (CCD-986Sk; ATCC) were cultured in medium composed of Iscove’s Modified Dulbecco′s Medium (Welgene, Gyeongsan, Korea), fetal bovine serum (Gibco^TM, Thermo Fisher Scientific, Rockford, IL, USA), and 1% penicillin/streptomycin (Welgene). Cells were also maintained at 37 °C and 5% CO₂.

MSC were induced to differentiate into adipogenic and osteogenic lineages, as described previously with minor modifications [49 (link)]. For osteogenic differentiation, MSCs were plated at 1 × 10⁵ cells/well in six-well culture plates. After 1 day of incubation, the cells were washed in PBS and then cultured in Dulbecco’s modified Eagle medium (DMEM; WELGENE, Daegu, Korea) supplemented with 0.1 μmol/L of dexamethasone, 100 μmol/L of L-ascorbic acid 2-phosphate, 10 mmol/L of β-glycerol phosphate, and 10% FBS. For adipogenic differentiation, MSCs were plated at 5 × 10⁵ cells/well in six-well culture plates. After 1 day of incubation, the cells were washed in PBS and then cultured in Iscove’s modified Dulbecco’s medium (WELGENE) supplemented with 1 μmol/L of dexamethasone, 0.2 mmol/L of indomethacin, 0.5 mmol/L of 3-isobutyl-1-methylxanthine, and 10% FBS. To examine the effect of PBM on MSC differentiation, treatment was performed once per day for 14 days of induction. After the induction period, differentiation was evaluated using Oil Red O staining for adipogenesis and Alizarin Red S staining for osteogenesis. Then, the degree of differentiation was quantified using a microplate reader. In addition, the expression of differentiation markers was also analyzed by real-time RT-PCR.

Human fibroblasts (CCD-986Sk; American Type Culture Collection, Manassas, VA, USA) were cultured in growth medium (Iscove’s modified Dulbecco’s medium; Welgene, Gyeongsan, Republic of Korea) supplemented with 10% fetal bovine serum (Gibco^TM, Thermo Fisher Scientific, Rockford, IL, USA) and 1% penicillin/streptomycin (Welgene) at 37 °C in an atmosphere containing 5% CO₂. To induce fibroblast senescence, the cells were treated with 350 μM H₂O₂ (Sigma-Aldrich, St. Louis, MO, USA) for 1.5 h, washed with Dulbecco’s phosphate-buffered saline (Gibco^TM, Thermo Fisher Scientific), and cultured in fresh growth medium for 72 h [53 (link)]. HIFU (LINEAR or DOT mode; 7 MHz frequency; 0.3, 0.5, or 0.7 J of energy) was applied, followed by incubation for an additional 48 h, after which cell lysates and supernatants were harvested for protein expression analyses.