Fig. 1. Effect of FCCP treatment on mitochondrial membrane potential and triglyceride content in differentiated adipocytes. (A) Determination of the m was assessed after differentiated (DIFF) adipocytes were not incubated or were incubated with 0.5 µM TMRE with or without 10 ng/ml TNF or 0.5 µM FCCP before FACS analysis. As positive control, differentiated cells were treated with 10 µM FCCP. (B,C) For TG determination, differentiated cells were incubated for 3 (white columns) or 6 days (black columns) with 10 ng/ml TNF or 0.5 µM FCCP. TG vesicles were then stained with ORO and the relative TG content in cell monolayers was (B) determined at 490 nm with a spectrophotometer and (C) visualized by phase-contrast microscopy (magnification 250x). (D) Glycerol release was measured in the 24-hour-old media using the INT kit, and (E) TG content was determined after lipid extraction in methanol-chloroform (using the same kit) in differentiated (DIFF) and undifferentiated (CTL) cells and then incubated for 1 (black columns), 3 (grey columns) or 6 (white columns) days with or without 10 ng/ml TNF, 0.5 µM FCCP or 10 µM isoproterenol (ISO). Representative results are expressed as percentages of differentiated cells. Values are means ± s.d. for n=3 experiments. ^***P<0.001, significantly different from DIFF. ^##P<0.01; ^###P<0.001, significantly different from DIFF+ISO.