Adipocyte Differentiation Assays

PPARγ2 activation in pre-adipocytes is required for the route of differentiation into mature adipocytes (Tontonoz et al. 1994). A key aspect of the process is the transformation of cells from spindly fibroblasts into spherical cells containing cytoplasmic fat globules that can imaged microscopically. Fibroblasts can be differentiated in vitro in a manner that reenacts most of the features characteristic of adipogenesis in vivo. 3T3-L1 cells are a well-characterized mouse embryonic fibroblast (pre-adipocyte) cell line routinely used to study adipocyte differentiation. When induced, differentiation of 3T3-L1 cells into mature adipocytes occurs within 7–9 d.

3T3-L1 cells were differentiated using a standard methylisobutylxanthine, dexamethasone, insulin (MDI) differentiation protocol (Zebisch et al. 2012). The insulin, 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone reagents (at 1,000× stock solutions) were included in the adipocyte kits purchased from Cayman Chemical Company. Briefly, for microscopic imaging studies, 400μL of cells were plated in 8-well chamber slides at a density of 4×105cells/mL in MEM supplemented with 10% bovine serum. For quantitative adipogenesis assays, 4×104 cells were plated in 96-well plates in 100μL media. The cells were allowed to adhere overnight and either used the next day or allowed to grow an additional day until reaching 90% confluence. On experimental day 1, cells were incubated with MEM supplemented with 10% FBS, 1μM insulin, 250μM IBMX, and 0.1μM dexamethasone in the absence or presence of rosiglitazone, GW9662, and/or phytoestrogen ligands. On days 4 and 6, the cells were administered MEM supplemented with 10% FBS and insulin in the presence or absence of rosiglitazone, GW9662, and/or phytoestrogen ligands. For the imaging studies, on day 8 the cells were fixed and stained with Oil Red O according to the manufacturer’s instructions. All of the reagents used in the staining, as described below, were part of the adipocyte differentiation assay kit (Cayman Chemical Company). Briefly, the medium was aspirated and cells were incubated in lipid droplet assay fixative for 15 min. The cells were next incubated with a wash solution for 5 min and then repeated. The wells were allowed to dry completely before adding Oil Red O working solution for 20 min. The cells were then washed twice for 5 min, and coverslips were mounted. Slides were viewed and photographed at 200× magnification using a Zeiss Axio Scope.A1 light microscope. For the quantitative studies, on day 8 the cells were incubated with AdipoRed and Hoechst reagent and assayed according to the manufacturers’ protocols. Briefly, the medium was removed and each well was washed with phosphate buffered saline (PBS). The cells were fixed in 2% paraformaldehyde for 30 min, washed with PBS, and incubated with AdipoRed assay reagent; after 10 min, fluorescence was read (Ex485nm/Em535) using a Spectramax i3X plate reader. The cells were co-incubated for 30 min with the nuclear dye Hoechst 33342 and read at Ex330/Em470 in order to quantitate the DNA content to allow for correction for cell number variances between samples. Lipid accumulation values were calculated between treatments by normalizing lipid intensity values to Hoechst intensity. All assays were performed in quadruplicate replicates, and a minimum of three independent studies were performed in each instance.