title: Quantitative Measurement of Cholesterol in Cell Populations Using Flow Cytometry and Fluorescent Perfringolysin O.
abstract: Methods to quantify intracellular cholesterol are valuable for the study of its trafficking and storage in normal cells and in lysosomal storage disorders. Traditionally, cholesterol has been tracked using the small molecule, filipin. Filipin can be difficult to visualize and visualization can be cytotoxic as it requires UV illumination. Here we describe a method to measure cholesterol using a fluorescently labeled, mutant form of Perfringolysin O, a soluble protein toxin that binds cholesterol specifically. This approach has been used to measure the impact of NPC1 deficiency on lysosomal cholesterol levels and monitor the rescue of cholesterol export under conditions that reduce the thickness of the lysosomal glycocalyx.
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305-5307, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305-5307, USA.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Lee
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305-5307, USA. pfeffer@stanford.edu.
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Pfeffer
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry*; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D003412:Cricetulus; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D019276:Glycocalyx / Q000378:metabolism* / Q000473:pathology; D006367:HeLa Cells; D006460:Hemolysin Proteins / Q000737:chemistry*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D016464:Lysosomal Storage Diseases / Q000378:metabolism* / Q000473:pathology; D008247:Lysosomes / Q000378:metabolism* / Q000473:pathology; D008562:Membrane Glycoproteins / Q000378:metabolism; D000077296:Niemann-Pick C1 Protein
chemical_list: D001427:Bacterial Toxins; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; C020452:Clostridium perfringens theta-toxin; D002784:Cholesterol
keywords: Cholesterol; Lysosome; Niemann Pick type C; Perfringolysin O