Abstract
Lipid droplets (LDs), initially thought to be mere lipid storage structures, are highly dynamic organelles with complex functions that control cell fate and behavior. In recent years, their relevance as therapeutic targets for a wide array of human diseases has been well established. Consequently, efforts to develop tools to study them have intensified, including assays that can accurately track LD levels in clinically relevant cell-based models. We previously reported that LD accumulation destines podocytes for lipotoxicity and cell death in renal diseases of metabolic and nonmetabolic origin. We also showed that LD accumulation in those cells serves as both a marker for disease progression and as a therapeutic target. Here, we describe a robust phenotypic screening method, using differentiated human podocytes, for identifying small-molecule compounds that rescue podocytes from LD accumulation and lipotoxicity under cellular stress. Major assay advances include 1) the use of a solvatochromic dye to improve LD staining, reduce background noise, and improve detection accuracy, 2) use of confocal imaging to reduce vertical overlap of LDs and enable accurate counting, 3) combining membrane and cytoskeleton stains to improve cell segmentation in confocal mode, and 4) use of an optimized spot detection algorithm that requires minimal configuration per individual run. The assay is robust and yields a Z-factor that is consistently >0.5.
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Gurumani, M., Mallela, S.K., Varona, J., Merscher, S., Fornoni, A., Al-Ali, H. (2023). A Robust Phenotypic Screening Assay Utilizing Human Podocytes to Identify Agents that Modulate Lipid Droplets. In: Bhattacharya, S.K. (eds) Lipidomics. Methods in Molecular Biology, vol 2625. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2966-6_15
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DOI: https://doi.org/10.1007/978-1-0716-2966-6_15
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Publisher Name: Humana, New York, NY
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