Subcellular Fractionation of Hela Cells for Lysosome Enrichment Using a Continuous Percoll-Density Gradient

The enrichment of lysosomes is a useful way to study their structure and function. These dynamic vesicles can be enriched from cell cultures in a variety of ways including immunoprecipitation and fluorescence-activated organelle sorting. These methods are extremely precise but often require the tran...

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Bibliographic Details
Published in:Bio-protocol 2019-09, Vol.9 (18), p.e3362-e3362
Main Authors: Carosi, Julian M, Hattersley, Kathryn J, Cui, Yi, Yang, Zhe, Teasdale, Rohan D, Sargeant, Timothy J
Format: Article
Language:English
Subjects:
Biology
Methods
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Summary:The enrichment of lysosomes is a useful way to study their structure and function. These dynamic vesicles can be enriched from cell cultures in a variety of ways including immunoprecipitation and fluorescence-activated organelle sorting. These methods are extremely precise but often require the transfection and expression of an affinity or fluorophore-tagged lysosomal membrane protein. A simpler approach uses differential density of subcellular organelles, which are characteristic to a particular type of organelle. Separation of organelles along a density-gradient enables fractionation to enrich for specific organelles (such as lysosomes) in their native state. This protocol outlines an optimized method for enriching lysosomes from HeLa cells with a continuous density-gradient that contains Percoll. Gentle cell lysis and extraction conditions yield dense-fractions that are enriched with functional and intact lysosomes, which can be assayed in downstream analyses. This method is quick (conducted in less than 2 h after harvesting cells), and can be easily scaled and optimized for other cell types.
ISSN:2331-8325
2331-8325
DOI:10.21769/BioProtoc.3362