Methods for making and observing model lipid droplets

Summary The mechanisms by which the lipid droplet (LD) membrane is remodeled in concert with the activation of lipolysis incorporate a complex interplay of proteins, phospholipids, and neutral lipids. Model LDs (mLDs) provide an isolated, purified system for testing the mechanisms by which the droplet composition, size, shape, and tension affects triglyceride metabolism. Described here are methods of making and testing mLDs ranging from 0.1 to 40 µm diameter with known composition. Methods are described for imaging mLDs with high-resolution microscopy during buffer exchanges for the measurement of membrane binding, diffusion, and tension via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), pendant droplet tensiometry, and imaging flow cytometry. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes in triglyceride metabolism.