Quantifying Glucose Incorporation into Lipids

¹⁴C-glucose incorporation was performed as previously described (37 (link)). Differentiated sWACs were incubated for 12 h with growth medium supplemented with 0.1 µCi D[¹⁴C(U)]- glucose/ml (American Radiolabeled Chemicals, St. Louis, MO, USA). Thereafter, cells were washed 4 times with ice-cold PBS, and neutral lipids were extracted with hexane/isopropanol (3/2, v/v). Thin-layer chromatography was performed with hexane/diethylether/acetic acid (70/29/1, v/v/v) as mobile phase. Lipids were visualized with iodine vapor. Visible bands were cut out, transferred into scintillation cocktail, and incubated overnight. The incorporated radioactivity was measured by liquid scintillation counting in the Tri-Carb 2300TR (Hewlett-Packard, Palo Alto, CA, USA) or the LS6500 (Beckman Coulter, Brea, CA, USA) scintillation counter. Total glucose incorporation into each lipid class was calculated, and values were normalized to protein content.