Intracellular and Plasma Membrane Cholesterol Labeling and Quantification Using Filipin and GFP-D4

Methods in Molecular Biology
Léa P WilhelmFabien Alpy

Abstract

Cholesterol, a major component of biological membranes, is rapidly trafficked and unevenly distributed between organelles. Anomalies of intracellular cholesterol distribution are the hallmark of a number of lysosomal lipid storage disorders. A major methodological obstacle for studying cholesterol trafficking is tracing this molecule in situ. The use of fluorescent probes that specifically bind cholesterol allows the visualization and imaging of cellular cholesterol. Here, we describe a series of assays optimized for quantifying free cholesterol in cell populations and at the single cell level, both at the plasma membrane and inside cells. These methods use two fluorescent probes: the D4 fragment of perfringolysin O fused to GFP (GFP-D4) and the polyene macrolide filipin. First, we report a robust method for quantifying plasma membrane cholesterol by flow cytometry using the GFP-D4 probe. Second, to optically distinguish and quantify intracellular cholesterol accumulation, we have adapted the classical filipin cholesterol staining protocol. Indeed, we observed that treatment of living cells with methyl-β-cyclodextrin, a chemical known to extract cholesterol from the plasma membrane, improves the visualization of the intracellul...Continue Reading

Citations

May 15, 2020·Chemical & Pharmaceutical Bulletin·Dan WangQiang Zhang
Feb 13, 2020·Frontiers in Microbiology·Svyatoslav S SokolovDmitry A Knorre
Jun 13, 2021·Biochimica Et Biophysica Acta. Molecular and Cell Biology of Lipids·Valentin SchoopDoris Höglinger
Jul 6, 2021·Frontiers in Bioengineering and Biotechnology·Xiao LuGhassan S Kassab

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