Mesc adipogenesis kit

Adipocytes obtained after 21 days of mESC differentiation were processed for Oil Red O staining by using the reagents provided in the mESC adipogenesis kit (Millipore). Oil Red O specifically stains lipids such as triglycerides and cholesteryl oleate [25 (link)]. For adipocytes derived from 3T3-L1 cells, Oil Red O (Sigma) reconstituted in isopropanol was diluted 3 : 2 with distilled water (dH₂O). Cells in the culture dish were rinsed with 1X Dulbecco's Phosphate-Buffered Saline (DPBS) and fixed with 4% paraformaldehyde for 20 minutes and then rinsed with 1X DPBS. Oil Red O staining solution was added and the cells were allowed to stain for 20 minutes. Cells were rinsed 4 times with 1X DPBS. To determine lipid accumulation in the adipocytes, after aspirating the last 1X DPBS rinse, 0.25 mL dye extraction solution was added to each well to extract the Oil Red O dye from the stained lipids. The plate was allowed to shake on an orbital shaker for 30 minutes and the extracted dye was transferred to a 96-well plate, and optical density (OD) was measured at 520 nm in a plate reader (Molecular Devices).

Adipogenesis was performed by using the reagents and protocol from a mESC adipogenesis kit (Millipore). Wild type TC-1 and Men1-KO-3.2N mESCs [17 (link)] were cultured on a feeder layer of primary mouse embryonic fibroblasts (MEFs) (ATCC) in ESC maintenance medium containing leukemia inhibitory factor (LIF). The mESCs were processed to obtain feeder-free cells by culturing on tissue culture treated dishes few times for an hour each to get rid of the attached feeder layer cells, and the unattached feeder-free mESCs were subsequently cultured on gelatin-coated dishes in ESC maintenance medium containing LIF. Cells were detached with accutase and plated in embryoid body (EB) formation medium (EB medium) (ESC maintenance medium without LIF) in nonadherent dishes for 2 days and then in medium containing inducer-A solution (all-trans-retinoic acid) for 3 days with a medium change every day. On day 5, the retinoic acid induced EBs were plated in gelatin-coated 24-well plates in adipocyte differentiation medium (EB medium + 3,3′,5-triiodo-L-thyronine (T3) + insulin). The adipocyte differentiation medium was changed every 2 days for a total of 21 days to allow adipocyte formation.