Progress in Drug Discovery & Biomedical Science Methods Article 1 A reliable and reproducible assay for determining the effect of natu- ral product on macrophages lipid uptake and cholesterol effux: A case study of maslinic acid Bee Kee Ooi 1 , Nafees Ahemad 2 , and Wei Hsum Yap 1* 1 School of Biosciences, Taylor’s University, Subang Jaya, Selangor, Malaysia. 2 School of Pharmacy, Monash University Malaysia, Petaling Jaya, Selangor, Malaysia Abstract : Macrophage foam cell formation represents a key feature that contributes to the development of atherosclerotic lesions. Assessment of cardioprotective natural compounds targeting macrophage foam cell formation processes including lipid uptake and cholesterol effux could lead to the identifcation of potential lead compounds for development into novel anti-atherosclerotic drugs. In this case study, maslinic acid, a natural product was used to study the effect on lipid uptake and cholesterol effux in THP-1-derived macrophages. Oil red O (ORO) staining and 1,1’-dioctadecyl-3,3,3’,3’-tetramethylin- docarbocyanine perchlorate-labeled oxidized low-density lipoprotein (Dil-labeled oxLDL) uptake assays were performed to determine lipid uptake by macrophages while cholesterol effux was assessed using 3-hexanoyl-NBD labeled cholesterol. ORO-stained images were further analyzed using ImageJ analysis software to determine intracellular lipid droplets accumu- lation and fow cytometric analysis of median fuorescence intensity were obtained to quantify Dil-labeled oxLDL uptake by macrophages. Meanwhile, 3-hexanoyl-NBD labeled cholesterol uptake and effux from THP-1-derived macrophages were characterized. The fuorescence intensity values obtained from the medium and cell lysates were used to determine the cho- lesterol effux. The results have shown that incubation with maslinic acid suppressed oxLDL-induced macrophage foam cell formation which may be contributed from its effect in reducing lipid uptake and enhancing cholesterol effux. In conclusion, the optimized ORO staining, Dil-labeled oxLDL uptake, and fuorescent-labeled cholesterol effux assays provide reproduc- ible and reliable results for assessment of foam cells formation. Keywords: Low-density lipoprotein (LDL); Oil red O (ORO), 1,1′-Dioctadecyl-3,3,3,3′-tetramethylindocarbocyanide per- chlorate (Dil); 3-hexanoyl-NBD labeled cholesterol Received: 30 th July 2019 Accepted: 28 th August 2019 Published Online: 10 th September 2019 *Correspondence: Yap Wei Hsum, School of Biosci- ences, Taylor’s University, Subang Jaya, Selangor, Ma- laysia; weihsum.yap@taylors.edu.my Citation: Ooi BK, Ahemad N, and Yap WH. A reliable and reproducible assay for determining the effect of natural product on macrophages lipid uptake and cholesterol effux: A case study of maslinic acid. Prog Drug Discov Biomed Sci 2019; 2(1): a0000031 Introduction Foam cell formation represents one of the early hallmarks of atherosclerotic lesion, which may result from imbal- anced lipid uptake and impaired cholesterol effux in macrophages. Modifed low-density lipoprotein (LDL) is thought to act as an infammatory factor in evoking atherosclerosis [1,2] . LDL within the intima layer may un- dergo modifcations, including oxidation, glycation, desi- alylation, and acylation. The modifed LDL is recognized by macrophages scavenger receptors [3-6] . In contrast, the effux of cholesterol in macrophages can be mediated through reverse cholesterol transporters or by a passive aqueous diffusion process [6,7] . Therapeutic intervention targeting foam cell formation which focuses on decreasing lipid uptake while enhancing cholesterol effux in macro- phages are expected to retard atherosclerosis progression. LDL is a spherical shaped particle with a diameter of 18 - 25 nm and has a density range of 1.019 - 1.063 g/ml [8,9] . It can be obtained from commercial suppliers or iso- lated from human plasma by ultracentrifugation meth- ods [10-12] . In this experiment, LDL was isolated from human plasma by density gradient ultracentrifugation using Beckman Coulter Optima L-100XP with an SW40 Ti swinging-bucket rotor, and followed by overnight di- alysis at 4°C. Two conventional methods including Oil red O (ORO) staining [13] and 1,1’-dioctadecyl-3,3,3’,3’- tetramethylindocarbocyanine perchlorate-labeled oxi- dized low density lipoprotein (Dil-labeled oxLDL) uptake assay [13,14] , were used to assess foam cell forma- tion in macrophages. Meanwhile, several methods have been used to study cholesterol effux from macrophages. Radioisotope-labeled cholesterol method has been tradi- tionally used for measuring cholesterol effux. Howev- er, this method is labor-intensive, and radioisotope has short shelf-life after preparation. Therefore, fuorescent- labeled cholesterol (3-hexanoyl-NBD labeled choles- Copyright 2019 by Ooi BK, Ahemad N et al. and HH Publisher. Tis work under licensed under the Creative Commons Attribution- NonCommercial 4.0 International Lisence (CC-BY-NC 4.0)