Quantifying Adipocyte Lipolysis via Glycerol Release

Cells were plated in 96-well plates (10386612, Fisher Scientific). On day 10, the cell culture medium was exchanged to 100 μl phenol red-free DMEM/F12 (21041-033, Thermo Scientific), supplemented according to Supplementary Table 2. On day 12, the cell culture medium was collected for basal lipolysis and exchanged to 80 µl phenol red-free DMEM/F12 supplemented with 2% fatty acid free BSA (3117057001, Sigma-Aldrich) and 0.1 mg ml⁻¹ ascorbic acid (500074, Merck), with or without 1 µM isoproterenol (I5627-5G, Sigma-Aldrich) for 3 h, after which the medium was collected. Glycerol release into the culture medium was quantified to measure lipolysis^{55 (link)}. Then, 20 µl medium or glycerol standards (G7793-5ML, Sigma-Aldrich) was transferred to a 96-well plate (M5686-40EA, Sigma-Aldrich). A 100 µl mixture of Free Glycerol Reagent (F6428-40ML, Sigma-Aldrich) and Amplex Ultrared (10737474, Fisher Scientific) was added and incubated for 15 min at room temperature before measurement in a Varioskan microplate reader (Thermo Fisher Scientific) at Excitation/Emission 530/590 nm. Glycerol release was normalized to cell number.