Characterization of fluorescent NBD-cholesterol efflux in THP-1-derived macrophages

Abstract

Macrophage cholesterol efflux is important in maintaining cellular lipid homeostasis and preventing the formation of lipid-laden foam cells. Although radioactive [3H]-cholesterol is widely used as a tracer in cholesterol efflux assays, the lengthy and labor-intensive assay procedure, and the radioactivity disposal procedure limit the use of this assay for high-throughput screening. In the present study, a novel procedure using fluorescent N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-xholen-3β-ol (NBD)-cholesterol was developed as a substitute for [3H]cholesterol for the measurement of cholesterol efflux in THP-1-derived macrophages. NBD-cholesterol uptake and metabolism in the THP-1 cells were characterized using fluorescent microscopy and spectrophotometry. NBD-cholesterol distributed rapidly into the cell organelles, with the exception of the nucleus. The uptake of NBD-cholesterol in the THP-1 macrophages was concentration-and time-dependent, and reached a plateau following 4 h incubation. The present study subsequently investigated whether NBD-cholesterol efflux was correlated with [3H]-cholesterol efflux in THP-1 derived macrophages and in human peripheral blood mononuclear cells (PBMCs). The results demonstrated that the percentage of efflux of NBD-cholesterol in the THP-1 cells was significantly correlated with that of [3H]cholesterol, at various concentrations of HDL or apoA-1 as lipid acceptors (R2=0.876 for HDL; R2=0.837 for apoA-1; P<0.001). In the PBMCs, NBD-cholesterol efflux also correlated significantly with [3H]-cholesterol efflux (R2=0.887 for HDL; R2=0.872 for apoA-1; P<0.001). Furthermore, NBD-cholesterol efflux in the THP-1 cells exhibited a similar trend to that obseved in the PBMCs. In conclusion, the results of the present study suggested that fluorescent NBD-cholesterol can be used as a sensitive and specific probe in cholesterol efflux assays in THP-1-derived macrophages.