Establishment of an in vitro flow cytometric detection method for lipid-accumulated cells

Abstract

Eukaryotic cells can store and converse excess lipid to the cytosolic lipid droplets. Adipogenesis of preadipocytes has been often used to study the molecular basis and the effect of obesity drugs on fat cell conversion. Many methods were developed for the detection of the cytosolic lipid droplets as Nile red, BODIPY 493/503 (4, 4-difluoro-1, 3, 5, 7, 8-pentamethyl-4-bora-3a, 4adiaza-s-indacene), BODIPY 665/676, 1,6-diphenylhexatriene (DPH), DAPI, Hoechst, Sudan III, and Oil-red O. The differences in the spectral properties of these lipophilic dyes and their advantages of each are discussed. In this study, an in vitro flow cytometric detection method was established for the detection of lipid-accumulated cells. Commonly, the longer the period of adipogenic induction, the greater the quantity of lipid in fat cell can accumulate. Thus, to determine whether increasing the fat stored within a cell would result in the greater granularity. 3T3-L1 cells in culture were hormonally induced for adipogenesis. Then, these cells were dissociated and analyzed in a flow cytometer at 0, 5, and 10 days post-induction. After adipogenic induction, the cells had become increasingly heterogeneous in their cellular granularity. The cells containing greater granular structure were markedly increased, and this increase in granularity positively correlated with the time of the post-adipogenic induction. On the other analysis, the 0 and 10 days post adipogenic induction of 3T3-L1 cells were gated for 4 regions. The R1 region contains cells with a level of granularity similar to that seen in the control cells (non-adipogenic induction), whereas R2 to R4 regions contain cells with increasing granularity. According to all data, we have successfully established an in vitro flow cytometric detection method for the detection of lipid-accumulated cells. We wish this method will be applied on the research of obesity drugs and the design of therapeutic strategies for obesity in the future.