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Supplementary key words lipid droplets • methods • cholesterol and trafficking • fluorescence and confocal imagingAtherosclerosis is characterized by the recruitment of inflammatory cells into the intima, where excessive uptake of modified LDL particles, such as oxidized LDL, by monocytes and macrophages leads to foam cell formation (1), promoting the initiation of atherosclerotic lesions. Subsequent endothelial activation leads to continuous recruitment of circulating monocytes into the subintimal area, further increasing inflammatory activity of these lesions, leading to plaque progression and, eventually, plaque destabilization (2-4). Recently, it has been suggested that migration of circulating monocytes is not merely a reflection of local endothelial activation near atherogenic areas, but circulating monocytes may also display pro-adhesive properties in patients at increased cardiovascular risk (5). The factors leading to this systemic activation of circulating monocytes remain to be established. In dyslipidemic states, lipid accumulation is thought to contribute to the proadhesiveness of the circulating cells. In the postprandial state, lipids were found to be taken up by circulating monocytes, resulting in increased expression of the integrin CD11c, binding partner of vascular adhesion molecule-1 (VCAM-1) Abstract The inflammatory profile of circulating monocytes is an important biomarker for atherosclerotic plaque vulnerability. Recent research revealed that peripheral lipid uptake by monocytes alters their phenotype toward an inflammatory state and this coincides with an increased lipid droplet (LD) content. Determination of lipid content of circulating monocytes is, however, not very well established. Based on Nile Red (NR) neutral LD imaging, using confocal microscopy and computational analysis, we developed NR Quantifier (NRQ), a novel quantification method to assess LD content in monocytes. Circulating monocytes were isolated from blood and used for the NR staining procedure. In monocytes stained with NR, we clearly distinguished, based on 3D imaging, phospholipids and exclusively intracellular neutral lipids. Next, we developed and validated NRQ, a semi-automated quantification program that detects alterations in lipid accumulation. NRQ was able to detect LD alterations after ex vivo exposure of isolated monocytes to freshly isolated LDL in a time-and dose-dependent fashion. Finally, we validated NRQ in patients with familial hypercholesterolemia and obese subjects in pre-and postprandial state. In conclusion, NRQ is a suitable tool to detect even small differences in neutral LD content in circulating monocytes using NR staining.-Schnitzler, J. G., S. J. Bernelot Moens, F. Tiessens, G. J. Bakker, G. M. Dallinga-Thie, A. K. Groen, M. Nieuwdorp, E. S. G. Stroes, and J. Kroon. Nile Red Quantifier: a novel and quantitative tool to study lipid accumulation in patient-derived circulating monocytes using confocal microscopy J. Lipid Res.
