Vpc23019

3T3- L1 pre-adipocytes (ATTC) were grown and differentiated as previously described [29 (link)]. Briefly, 3T3-L1 pre-adipocytes were grown to confluence in DMEM (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin. Following 2 days post confluence and clonal expansion adipocyte differentiation was initiated by treating cells for 3 days with 5 µg/mL insulin and 1 µM dexamethasone alone or insulin + dexamethasone + 1 µM sphingosine 1-phosphate (Sigma). Thereafter, media was changed every 3 days with only 100 ng/ml insulin. In some experiments adipocyte differentiation was carried out in the presence or absence of the S1PR1/3 antogonist VPC23019 (1 µM; Sigma). Ten days post-differentiation the cells were fixed with 10% Zn formalin for 20 minutes and permeabilized with 100% propylene glycol for 3 minutes. Fixed cells were then stained with Oil Red O in 100% propylene glycol for 1 hour at 37°C and destained with 60% propylene glycol using gentle agitation for 1 minute. The Oil Red O extractions were done using two 500 µL aliquots of isopropanol combined and absorbance was measured spectrophotometrically at 510 nm.