Quantification of Endothelial Fatty Acid Uptake using Fluorescent Fatty Acid Analogs.

Endothelial cells (ECs) play a central role in regulating fatty acid (FA) transport from the bloodstream into metabolic tissues, yet tools to quantify EC FA uptake in a reliable, scalable manner remain limited. Here, we present a rapid, quantitative, and cost-effective assay to measure FA uptake in ECs using fluorescent FA analogs (BODIPY-C12 and BODIPY-C16), which allow investigation of chain length-specific uptake dynamics in a 96-well plate format. The protocol incorporates positive (3-hydroxyisobutyrate, lactate) and negative (niclosamide) controls and is validated in both primary (HUVECs) and immortalized (EA.hy926) EC lines. The assay detects time- and dose-dependent FA uptake, with results normalized to cell number using Hoechst nuclear staining and corrected for background using appropriate controls. It can be adapted to a variety of cell types, imaging modalities, and experimental conditions, including live-cell imaging and pulse-chase formats. Compared to traditional lipid staining or radiolabeled tracers, this method offers improved safety, speed, and versatility while capturing dynamic FA uptake in live cells. This assay provides a robust platform for studying the regulation of endothelial lipid transport and its contribution to metabolic disease.