4.6. Intracellular Lipid Measurement

THP-1 macrophages (1 × 10⁶) cultured under HG conditions and stimulated with oxLDL in the presence or absence of RA as described above were washed three times with ice-cold PBS and harvested in 50 µL of chloroform:isopropanol:NP-40 (200 mL, 7:11:0.1). The extract was transferred into a centrifuge tube and spun for 10 min at 15,000× g. The organic phase was transferred into a new tube and air-dried at 50 °C to remove the chloroform. The tube was then dried at 100 °C for 30 min to remove trace organic solvent. Total cholesterol and free cholesterol in the dried lipids were quantified using the Total Cholesterol and Cholesteryl Ester Colorimetric/Fluorometric Assay Kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s instructions. Cholesteryl ester was obtained by subtracting free cholesterol from total cholesterol. The cholesterol content in the sample was calculated from the following formula: sample cholesterol concentration (C) = B/V × D (µg/µL), where B is the amount of intracellular cholesterol (µg), V is the volume of sample added to the reaction well (µL) and D is the sample dilution factor.