title: Luminol electrochemiluminescence for the analysis of active cholesterol at the plasma membrane in single mammalian cells.
abstract: A luminol electrochemiluminescence assay was reported to analyze active cholesterol at the plasma membrane in single mammalian cells. The cellular membrane cholesterol was activated by the exposure of the cells to low ionic strength buffer or the inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT). The active membrane cholesterol was reacted with cholesterol oxidase in the solution to generate a peak concentration of hydrogen peroxide on the electrode surface, which induced a measurable luminol electrochemiluminescence. Further treatment of the active cells with mevastatin decreased the active membrane cholesterol resulting in a drop in luminance. No change in the intracellular calcium was observed in the presence of luminol and voltage, which indicated that our analysis process might not interrupt the intracellular cholesterol trafficking. Single cell analysis was performed by placing a pinhole below the electrode so that only one cell was exposed to the photomultiplier tube (PMT). Twelve single cells were analyzed individually, and a large deviation on luminance ratio observed exhibited the cell heterogeneity on the active membrane cholesterol. The smaller deviation on ACAT/HMGCoA inhibited cells than ACAT inhibited cells suggested different inhibition efficiency for sandoz 58035 and mevastatin. The new information obtained from single cell analysis might provide a new insight on the study of intracellular cholesterol trafficking.
authors.affiliation: Key State Labortorary of Analytical Chemistry for Life Science and School of Chemistry and Chemical Engineering, Nanjing University, Jiangsu, 210093, China.
authors.forename: Guangzhong
authors.initials: G
authors.lastname: Ma
authors.forename: Junyu
authors.initials: J
authors.lastname: Zhou
authors.forename: Chunxiu
authors.initials: C
authors.lastname: Tian
authors.forename: Dechen
authors.initials: D
authors.lastname: Jiang
authors.forename: Danjun
authors.initials: D
authors.lastname: Fang
authors.forename: Hongyuan
authors.initials: H
authors.lastname: Chen
mesh_terms: D000214:Acyl Coenzyme A / Q000037:antagonists & inhibitors; D000577:Amides / Q000494:pharmacology; D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002784:Cholesterol / Q000032:analysis* / Q000378:metabolism; D002789:Cholesterol Oxidase / Q000737:chemistry; D055664:Electrochemical Techniques; D004566:Electrodes; D006861:Hydrogen Peroxide / Q000737:chemistry; D008148:Lovastatin / Q000031:analogs & derivatives / Q000494:pharmacology; D008163:Luminescent Measurements; D008165:Luminol / Q000737:chemistry; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D017646:Organosilicon Compounds / Q000494:pharmacology; D059010:Single-Cell Analysis / Q000379:methods*; D002785:Sterol O-Acyltransferase / Q000037:antagonists & inhibitors
chemical_list: D000214:Acyl Coenzyme A; D000577:Amides; D000924:Anticholesteremic Agents; D017646:Organosilicon Compounds; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; C012258:mevastatin; D008165:Luminol; C040256:SAN 58035; D002784:Cholesterol; D008148:Lovastatin; D006861:Hydrogen Peroxide; D002789:Cholesterol Oxidase; D002785:Sterol O-Acyltransferase