Quantification of lipid in cultured 3T3-L1 adipocytes

A preliminary study was conducted to quantify the lipid produced by differe ntiated 3T3-L1 cells after incubation in Dulbecco's Modified Eagle Medium ( DMEM) containing 10% foetal bovine serum (FBS), supplemented with or withou t dimethyl-sulphoxide (Dh ISO; 9.6 gl/l) and acetone (1.2 g/l). The two med ia treatments were applied to 3T3-L1 cells, plated at either 15K or 30K cel ls per well in 24-well plates. Cells were grown to confluence (96 h) and th en treated with dexamethasone, methyl-isobutylxanthine and insulin for 48h and later maintained in their respective media treatments for another 144 h . Cells from each treatment were recovered after two, 5-min incubations wit h trypsin, washed and resuspended in DMEM and counted on a haemocytometer. The lipid in the cells was extracted with hexane derivatized with tetrameth yl-guanidine and analysed by gas chromatography. Final mean cell density wa s 68 (s.e. 0.18) X 10(5) and 4.6 (s.e. 0.19) X 10(5) when initially plated at 30K and 15K cells per well, respectively. Inclusion of DMSO and acetone in the medium did not affect final cell numbers. Plating density did not af fect concentration of lipid (0.55 (s.e. 0.08) mg per I X 105 cells) but inc lusion of DMSO and acetone led to overall decreases in total lipid concentr ation. Results indicate that initial plating density influenced final cell number in treatment cultures, but that DMSO and acetone treatments only had an effect on final lipid concentration. Collectively these data suggest th at the application of treatments to cell cultures may be influenced by the carrier vehicle that the treatment is contained in and this should be consi dered when developing an in vitro system to evaluate growth and development adipocytes.