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 intracellular cholesterol pool with filipin. To complement these staining procedures, we developed an image analysis protocol based on image segmentation to quantify, in a robust manner, intracellular cholesterol stained with filipin. Thus, this chapter is a guideline for cellular cholesterol staining and signal quantification.
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Acknowledgments
We thank Thomas Di Mattia and Alastair McEwen for their critical reading of the manuscript. We thank the members of the Molecular and Cellular Biology of Breast Cancer team (IGBMC) for helpful advice and discussions, and the IGBMC imaging center. L.V. received an allocation from the Ministère de l’Enseignement Supérieur et de la Recherche (France; http://www.enseignementsup-recherche.gouv.fr/) and L.P.W. a fellowship from the Fondation pour la Recherche Médicale (https://www.frm.org/). This work was supported by grants from the Institut National Du Cancer INCA (INCA_9269; www.e-cancer.fr), the Ligue Contre le Cancer (Conférence de Coordination Interrégionale du Grand Est; https://www.ligue-cancer.net), The Ara Parseghian Medical Research Fund (http://parseghianfund.nd.edu/), Vaincre les maladies lysosomales (http://www.vml-asso.org/) and the grant ANR-10-LABX-0030-INRT, a French State fund managed by the Agence Nationale de la Recherche under the frame program Investissements d’Avenir ANR-10-IDEX-0002-02. We also acknowledge funds from the Institut National de Santé et de Recherche Médicale (http://www.inserm.fr/), the Centre National de la Recherche Scientifique (http://www.cnrs.fr/), and the Université de Strasbourg (http://www.unistra.fr).
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Wilhelm, L.P., Voilquin, L., Kobayashi, T., Tomasetto, C., Alpy, F. (2019). Intracellular and Plasma Membrane Cholesterol Labeling and Quantification Using Filipin and GFP-D4. In: Drin, G. (eds) Intracellular Lipid Transport. Methods in Molecular Biology, vol 1949. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9136-5_11
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DOI: https://doi.org/10.1007/978-1-4939-9136-5_11
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