2.1. Culture of Primary Subcutaneous Adipocytes

Primary human preadipocytes were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA). Cells were cultured for 48 h in Preadipocyte Medium (PAM) which contains Dulbecco’s Modified Eagle Medium (DMEM)/Ham’s F-12 (1:1, v/v), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 5% fetal bovine serum (FBS), penicillin, streptomycin, and amphotericin B. Cells were maintained at 37 °C and 5% CO₂ in a humidified incubator. For cell differentiation into mature adipocytes, cells were cultured for 12 days in Preadipocyte Differentiation Medium (PADM) that contains DMEM/Ham’s F-12 (1:1, v/v), HEPES pH 7.4, 5% FBS, biotin, pantothenate, human insulin, dexamethasone 3-isobutyl-1-methylxanthine (IBMX) peroxisome proliferator-activated receptor gamma (PPARγ) agonist, penicillin, streptomycin, and amphotericin B. Finally, cells were maintained in differentiation maintenance medium. This medium consists of DMEM/Ham’s F-12 (1:1, v/v), HEPES pH 7.4, 5% FBS, biotin, pantothenate, human insulin, dexamethasone, penicillin, streptomycin, and amphotericin B. All cell culture media were purchased from ScienCell Research Laboratories. To confirm differentiation, cells were stained with Oil Red O (BioVision Inc., Milpitas, CA, USA). Briefly, after washing with 1X PBS, cells were fixed in 10% formalin for 1 h, washed with distilled water, and incubated with 60% isopropanol for 5 min followed by Oil Red O Stain for 10 min. After stain removal, cells were washed with distilled water until the excess stain was completely removed. For counterstaining, cells were incubated in hematoxylin for 60 s, washed several times with distilled water, and imaged with an inverted microscope (Olympus, Tokyo, Japan). Differentiated cells (at day 12) showed cytoplasmic accumulations of fat globules compared to the undifferentiated state on day 0 (Figure 1A). Also, mRNA levels of AdipoQ and FABP4 genes were measured via Real-time PCR (described below). Both genes were significantly upregulated in differentiated adipocytes (Figure 1B).

Assessment of adipocyte differentiation. (A) Microscopic images that demonstrate cytoplasmic accumulation of lipid droplets in adipocytes by differentiation days 0 and 12. (B) Quantitative measurements of FABP4 and AdipoQ mRNA levels in differentiated adipocytes using real-time PCR. Results represent the means ± SD for three independent experiments, (* p < 0.05) for comparing day 12 with day 0.