Abstract
Macrophage-derived foam cell formation elicited by oxidized low-density lipoprotein (oxLDL) is the hallmark of early atherogenesis. Detection of foam cell formation is conventionally practiced by Oil Red O (ORO) staining of lipid-laden macrophages. Other methods include 1,1′-dioctadecyl-3,3,3′3′-tetra-methylindocyanide percholorate (DiI)-labeled oxLDL (DiI-oxLDL) uptake and Nile Red staining. The purpose of the present study is to report an optimized method for assessing foam cell formation in cultured macrophages by ORO staining and DiI-oxLDL uptake. After incubation with oxLDL (50 μg/ml) for 24 h, the macrophages were fixed, stained with ORO for just 1 min, pronounced lipid droplets were clearly observed in more than 90% of the macrophages. To test the in vivo applicability of this method, lesions (or foam cells) of cryosections of aortic sinus or primary mouse peritoneal macrophages from ApoE deficient mice fed a high cholesterol diet were successfully stained. In another set of experiments, treatment of macrophages with DiI-oxLDL (10 μg/ml) for 4 h resulted in significant increase in oxLDL uptake in macrophages as demonstrated by confocol microscopy and flow cytometry. We conclude that the optimized ORO staining and fluorescent labeled oxLDL uptake techniques are very useful for assessing intracellular lipid accumulation in macrophages that are simpler and more rapid than currently used methods.
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Acknowledgments
This study was supported by research grants from National Natural Science Foundation of China (No: 30472022), National Major Project “Key New Drug Creation and Manufacturing Program” (No: 2009ZX09102-152, 2009ZX09303-007). The authors would like to gratefully acknowledge the skillful technical assistance of Dr. Jing-Hua Ou, Dr. Xiao-Lu Duan for help with confocol and fluorescent microscopy. FACS was performed at the Flow Cytometry Core Facility of School of Life Sciences, Sun Yat-sen University (Guangzhou, China). We would like to extend our gratitude to all members of Department of Pharmacology and Toxicology for technical assistance and helpful discussions. Suowen Xu received “New Investigator Awards” from Ministry of Education of the People’s Republic of China.
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Suowen Xu and Yan Huang contributed equally to this work.
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Xu, S., Huang, Y., Xie, Y. et al. Evaluation of foam cell formation in cultured macrophages: an improved method with Oil Red O staining and DiI-oxLDL uptake. Cytotechnology 62, 473–481 (2010). https://doi.org/10.1007/s10616-010-9290-0
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DOI: https://doi.org/10.1007/s10616-010-9290-0