Quantitative and qualitative methods using fluorescence microscopy for the study of modified low density lipoproteins uptake.
Atherosclerosis and heart disease are the main cause of death in United States. The development of atherosclerosis includes lipid deposition and foam cell formation in the artery wall. Scavenger Receptors A-I and II (SRA-I/II) have an important role of in foam cell formation and atherogenesis. Most of the SRA-I/II studies had been performed using Iodine-125-radiolabeled modified low-density lipoprotein. This report attempts to validate the use of fluorescence microscopy techniques as an alternative to obtain qualitative and quantitative information of the uptake of fluorescence-labeled acetylated low-density lipoprotein (AcLDL) in adherent CHO cells expressing SRA-I/II. After verifying the protein expression of SRA-I and II, uptake was quantified using a Laser Scan Cytometer, and images of cells containing fluorescent AcLDL were obtained. A significant increase in fluorescence was found in the cells transfected with SRA-I/II vs. those with empty vector. When SRA-I/II competitive ligands were used, the uptake of AcLDL was significantly decreased. In conclusion, the use of fluorescence microscopy techniques in obtaining qualitative and quantitative information of the uptake of fluorescence-labeled AcLDL by adherent cells, such as CHO cells, is an alternative to the traditional use of radiolabeled iodine.
Leyva
Francisco J
FJ
Division of Lung Diseases, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA. leyvaf@mail.nih.gov
Pershouse
Mark A
MA
D000107:Acetylation; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D051116:Receptors, Scavenger / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL; D051116:Receptors, Scavenger