Oil red o

Freshly sorted cells were expanded as described above. After 5–7 days, medium was changed to adipogenic induction medium consisting of DMEM + 10% FBS + 1% penicillin/streptomycin + 1mM Dexamethasone (Sigma) + 100nM Insulin (Sigma) + 1mM Roziglitazone (Cayman Chemical) + 0.5 mM 3-isobutyl-1-methylxanthine (Sigma) for 2 days, and then replaced with adipogenic differentiation medium consisting of DMEM + 10% FBS + 1% penicillin/streptomycin + 100nM Insulin. Cells were kept in differentiation medium for 6 days, and then fixed with 4% Paraformaldehyde and stained with Oil Red O (Sigma) for one hour at room temperature [11 (link)]. Oil Red O staining of lipid droplets within adipocytes was analyzed by standard microscopy using an Olympus IX51 inverted microscope.

3T3-L1 cells were cultured in 60 mm diameter dishes (8.4×10⁴ cells/dish) and treated with NNMT inhibitor dissolved in culture media with/without adipogenic factors (1 mM IBMX, 1 µM dexamethasone, 10 µg/ml of insulin) during each of the scheduled media changes during the differentiation process (described above). On day 9 post-differentiation, cells were subjected to quantitative oil red O (Thermo Fisher Scientific; Waltham, MA, USA) staining as adapted and modified from published protocols.[12 (link)] Briefly, cells were washed twice with PBS, fixed with 10% formalin for 30 min at room temperature, and stained with oil red O working solution (~0.2% oil red O in 99% isopropanol) for 30 min. Cells were then washed five times or with sterile water until unincorporated oil red O stain was completely removed. Images of oil red O staining in control and inhibitor-treated cells were digitally photographed using a light microscope (Olympus BX41; Tokyo, Japan). After image capture, 2-propanol (3.5 mL) was added to each dish for 10 min to dissolve the oil red O stain and absorbance was quantified in a plate reader set at 492 nm wavelength. To ensure the absorbance from oil red O staining was within the linear detection range of the plate reader, a calibration curve was established for oil red O staining in adipocytes using a previously described protocol.[23 (link)]