Pulse-Chase Analysis of Lipid Dynamics

The procedure for the pulse-chase analysis of the S2 cells was modified from Miyata [27 (link)] and Tamura [54 (link)]. The S2 cells were incubated with NBD-PA, NBD-PC or NBD-PE (Avanti Polar Lipids, Inc.) for 20 min. The S2 cells were washed, then incubated for different periods of time. After harvesting, the total lipids in the cells were extracted and resuspended in chloroform/methanol (1:2, vol/vol). The lipid samples were separated by TLC on silica gel 60 F₂₅₄ plates (Merck,1.05729.0001) using a solvent system of chloroform/methanol/25% ammonia, 65:35:5 [26 (link)]. The TLC plates were detected with a Typhoon 9500 imager and the images were analyzed with Image J (1.51j8).