AimIn this study, fluorescence labeled cholesterol (NBD-cholesterol) was used to establish a new method to evaluate the cholesterol efflux, and compared NBD-cholesterol with ³H-cholesterol in assay of cholesterol efflux.MethodsIsolated human peripheral blood monocyte cells from volunteers were differentiated into human macrophages.Human monocyte line THP-1 and mouse monocyte line J774 cells were cultured in vitro.THP-1 macrophages were incubated with different concentrations of NBD-cholesterol to choose an optimistic concentration and incubation time.NBD-cholesterol efflux rate was evaluated from three different kinds of macrophages.NBD-cholesterol uptake and efflux rate were reflected by fluorescent intensity measurement by multi-label counter.The NBD-cholesterol efflux capacity in human macrophages was estimated by the correlation to ³H-cholesterol efflux in human macrophages.ResultsNBD-cholesterol distributed rapidly into cells organelles except nucleus.Uptake of NBD-cholesterol in THP-1 macrophages was concentration dependent, but time to plateau was not dependent on concentration.NBD-cholesterol efflux rate reflected an appropriate cholesterol efflux rate in human macrophages, THP-1 macrophages and mouse macrophages.Both apolipoprotein A1 mediated and HDL mediated ³H-cholesterol efflux rates were significant correlated to NBD-cholesterol efflux rate in human macrophages (r=0.968, p<0.001).ConclusionNBD-cholesterol was a useful, sensitive and environmental protective probe for assay of human macrophage cholesterol efflux rate.