Extraction and quantification of lipids in cells and media

Lipids were extracted from C3H10T1/2 cells and culture media using the Folch method (55) with minor modifications. Briefly, whole culture medium (1 ml) from each well of a 12-well plate was collected in a separate tube. Cells were washed with phosphate buffered saline (PBS) and collected in 1 ml PBS and homogenized. The media or cell homogenate was mixed in 5 ml of chloroform:methanol (2:1, vol/vol) by shaking vigorously several times and centrifuged at 2,500 g for 15 min. The bottom organic layer was transferred to a new glass tube. The remaining aqueous phase and interphase including the soluble protein were mixed with 5 ml chloroform by vigorous shaking, followed by centrifugation at 2,500 g for 15 min. The bottom organic layer was combined with the first collected organic layer. The combined organic phase was evaporated using nitrogen, and then the dried lipids were resuspended in 0.5% Triton X-100 in water. Samples were stored in −80°C until lipid analysis. TG, TC, unesterified cholesterol (UC), and phospholipid levels in lipid extractions from cells and from culture media were measured separately using a colorimetric assay at the UCLA GTM Mouse Transfer Core (56). Intracellular lipids were normalized to the cellular protein amount measured by BCA protein assay kit (Pierce, Rockford, IL). Extracellular lipids are presented as lipid quantity in 1 ml of collected media.