Background

Lipid droplets (LDs) are unique organelles with characteristic functions. Different from common cellular organelles, each LD is defined by a monolayer of phospholipids, which encloses a core of neutral lipids such as triacylglycerol (TAG), also known as triglyceride, and sterol esters. As storage organelles, LDs play a major role in the homeostasis of lipids and energy, as well as other cellular events. By storing excess fatty acids in the form of TAG, LDs prevent lipotoxicity and protect cells against oxidative stress. Importantly, LDs make contacts with virtually all other organelles and regulate critical cellular processes, including membrane trafficking, protein turnover and viral infection (Olzmann and Carvalho, 2018; Gao et al., 2019). Moreover, the LD surface is decorated with various proteins that are involved in the synthesis and hydrolysis of TAG, and in the transport of lipids between LDs and other organelles (Kory et al., 2016; Kumar et al., 2018; Du et al., 2020).

Eukaryotic cells have well-established pathways to synthesize TAG stored in LDs (Coleman and Mashek, 2011). Studies on the formation and growth of LDs often involve the extraction of TAG from cells or tissues for quantification. Some sensitive methods, such as LC-MS, are available for this purpose, but requiring expensive equipment and being time-consuming. Commercially available lipid quantification kit, on the other hand, can provide a quick, accurate, and economical method to measure cellular levels of TAG. In this protocol, we describe a reliable method using a fluorometric lipid quantification kit to measure TAG extracted from HeLa cells that were grown in the presence of oleic acid to induce LD formation. The lipid quantification kit takes advantage of a lipid binding molecule that emits bright fluorescence only when bound to extracted TAG. Thus, the amount of TAG can be readily quantified by a simple fluorescence readout.