Objectives Macrophage cholesterol efflux plays an important role in maintaining cellular lipid homeostasis, and preventing cells from formation of lipid-laden foam cells. Although radioactive [3H]-cholesterol was widely used as a tracer in cholesterol efflux assay, time- and labour-consuming assay procedure and radioactivity disposal procedure may limit its use in high-throughput screening. Here, we developed a new method using fluorescent NBD-cholesterol as a substitute for [3H]-cholesterol to measure cholesterol efflux in THP-1 derived macrophages.
Methods THP-1 cells were cultured in RPMI 1640 with 20% FBS, and differentiated into macrophages under incubation with 100 ng/ml of phorbol myristate acetate (PMA) for 72 h. NBD-cholesterol uptake and metabolism in THP-1 derived macrophages were characterised using fluorescent microscope and spectrophotometer. Cholesterol efflux in THP-1 derived macrophages was measured using either 22-NBD-cholesterol or [3H]-cholesterol as a tracer. The correlation data was obtained after compared percentage efflux of NBD-cholesterol with that of [3H]-cholesterol. NBD-cholesterol efflux was also measured in THP-1 cells compared with human peripheral blood mononuclear cells (PBMCs).
Results NBD-cholesterol distributed rapidly into cell organelles except nucleus. Uptake of NBD-cholesterol in THP-1 macrophages was concentration- and time-dependent, and reached a plateau after 4-h incubation. Next, we measured cholesterol efflux in THP-1 derived macrophages using either 22-NBD-cholesterol or [3H]-cholesterol as a tracer. The correlation data was obtained after compared percentage efflux of NBD-cholesterol with that of [3H]-cholesterol. Our results showed that percentage efflux of NBD-cholesterol was significantly correlated to that of [3H]-cholesterol using either apoA-1 or HDL as lipid acceptor (R2=0.882 for apoA-1, and R2=0.887 for HDL, respectively, p<0.001). Furthermore, NBD-cholesterol efflux in THP-1 cells showed similar trend with that in human peripheral blood mononuclear cells (PBMCs).
Conclusions Fluorescent NBD-cholesterol can be used as a sensitive and specific probe for cholesterol efflux assay in THP-1 derived macrophages.