Selection of clonal preadipogenic cell line as a rapidly differentiating adipogenesis model.

<p>(A) Schematic display of robust OP9 adipogenesis demonstrating the desired morphological characteristics of a rounded cell, peripheral nucleus, lipid accumulation, and a coalesced lipid droplet. Triglyceride accumulation shown in red and nucleus shown in blue. OP9 cells differentiate over the course of 72 hours. After one day of insulin oleate (IO) media exposure, cells begin to accumulate small lipid droplets. After two days of IO media exposure, cells adopt a round morphology, the nucleus migrates to the periphery, and the cytoplasm is filled with lipid droplet. After three days of IO media exposure, cells resemble mature adipocytes. The nucleus is peripherally located and lipid droplets have fused into one large lipid droplet. OP9 cells are capable of differentiation at sub-confluent levels. (B) OP9 clonal populations were differentiated into adipocytes using insulin oleate (IO) media. Clone K (orange box) demonstrates desired adipogenesis morphology. Cells were fixed with paraformaldehyde and then stained with the lipophilic dye, Nile Red. Nile Red stain is shown in red. The arrow points to a mature adipocyte. Magnification is 40X. (C) Triglyceride (TG) content of clonal OP9 cell lines after 72 hours exposure to IO media, low serum media (lsm), or propagation media (pm), using TG assay. Assays performed in triplicate. Clone K (orange box) robustly accumulates triglyceride. Clone K consistently demonstrated adipocyte morphology of a rounded cell, coalesced lipid droplet, and triglyceride accumulation. Therefore, clone K OP9 cells were selected for further study.