ArticleMethods in molecular biology (Clifton, N.J.)2025
Methods for Visualizing and Quantifying Cholesterol Distribution in Mammalian Cells Using Filipin and D4 Probes.
Article in Methods in molecular biology (Clifton, N.J.), 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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4 citing papers in PubMed.
- Fatty acid synthesis therapy-induced senescence (FASTIS) in cancer cells.Cell death & disease · 2026Article
- Lysosomal lipid metabolism promotes tumor cell invasion through local energetics and membrane lipid remodeling.bioRxiv : the preprint server for biology · 2026Article
- Live-Cell Imaging and Lipidomics of Low-Density Lipoprotein Containing Intrinsically Fluorescent Cholesteryl Esters.Analytical chemistry · 2026Article
- Src-dependent tyrosine-phosphorylation of NM2A has a protective role against bacterial pore-forming toxins.PLoS pathogens · 2026Article
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7 authors.
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Abstract
Cholesterol is a key component of biological membranes and, like many cellular lipids, is unevenly distributed among organelles. Disruptions in cholesterol trafficking are associated with various pathologies, including lysosomal lipid storage disorders, often characterized by intracellular cholesterol accumulation. A significant challenge in studying cholesterol trafficking is the lack of easy methods to trace this molecule in situ. Fluorescent probes that specifically bind cholesterol have enabled the visualization and imaging of cholesterol distribution within cells. This chapter details optimized methods for visualizing and quantifying free cholesterol at the plasma membrane and intracellulaly, both in individual cells and in large cell populations. These methods use two fluorescent probes: the D4 fragment of perfringolysin O fused to monomeric EGFP (mEGFP-D4 and the more sensitive mutant mEGFP-D4H) and the polyene macrolide filipin. We describe robust methods for quantifying plasma membrane cholesterol by flow cytometry and to visualize intracellular cholesterol pools by light microscopy. Furthermore, we introduce a refined filipin staining protocol that enhances intracellular cholesterol detection. For precise quantification, we developed an automated image analysis pipeline. This chapter provides a comprehensive guide for staining and quantifying cellular cholesterol, offering valuable tools for studying cholesterol dynamics in mammalian cells.
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