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Quantification of lipid in cultured 3T3-L1 adipocytes

Published online by Cambridge University Press:  18 August 2016

P. S. Mir ,
J. L. Vierck ,
Z. Mir  and
M. V. Dodson
P. S. Mir
Affiliation:
Agriculture and Agri-Food Canada Research Centre, Lethbridge, AB, Canada
J. L. Vierck
Affiliation:
Department of Animal Sciences, Washington State University, Pullman, WA, USA
Z. Mir
Affiliation:
Agriculture and Agri-Food Canada Research Centre, Lethbridge, AB, Canada
M. V. Dodson
Affiliation:
Department of Animal Sciences, Washington State University, Pullman, WA, USA
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Abstract

A preliminary study was conducted to quantify the lipid produced by differentiated 3T3-L1 cells after incubation in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% foetal bovine serum (FBS), supplemented with or without dimethyl-sulphoxide (DMSO; 9•6 g/l) and acetone (1•2 g/l). The two media treatments were applied to 3T3-L1 cells, plated at either 15K or 30K cells per well in 24-well plates. Cells were grown to confluence (96 h) and then treated with dexamethasone, methyl-isobutylxanthine and insulin for 48 h and later maintained in their respective media treatments for another 144 h. Cells from each treatment were recovered after two, 5-min incubations with trypsin, washed and resuspended in DMEM and counted on a haemocytometer. The lipid in the cells was extracted with hexane derivatized with tetramethyl-guanidine and analysed by gas chromatography. Final mean cell density was 6•8 (s.e. 0•18) ✕ 105 and 4•6 (s.e. 0•19) ✕ 105 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 affect concentration of lipid (0•55 (s.e. 0•08) mg per 1 ✕ 105 cells) but inclusion of DMSO and acetone led to overall decreases in total lipid concentration. 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 that the application of treatments to cell cultures may be influenced by the carrier vehicle that the treatment is contained in and this should be considered when developing an in vitro system to evaluate growth and development of adipocytes.

Keywords

adipocytesgas chromatographyhaemocytometerplating densitylipids
Type
Research Article
Information
Animal Science , Volume 71 , Issue 3 , December 2000 , pp. 521 - 526
Copyright
Copyright © British Society of Animal Science 2000

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