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. 2025 Sep 19;6(3):103987.
doi: 10.1016/j.xpro.2025.103987. Epub 2025 Jul 26.

Protocol to isolate stress granules in HeLa cells using fluorescence-activated non-membrane condensate isolation

Yilong Zhou  1 , Maria Shvedunova  2 , Asifa Akhtar  3
Affiliations

Protocol to isolate stress granules in HeLa cells using fluorescence-activated non-membrane condensate isolation

Yilong Zhou et al. STAR Protoc. .

Abstract

Various stress stimuli induce cytosolic stress granules (SGs) in mammalian cells, which are composed of RNA and RNA-binding proteins and have important physiological functions. Here, we present a protocol for isolating SGs using fluorescence-activated non-membrane condensate isolation (FANCI). We describe steps for seeding and stressing G3BP1-mCherry HeLa cells. We then detail procedures for purifying SGs by FANCI with flow cytometry. This protocol has potential application in studying SGs from other cells in the future. For complete details on the use and execution of this protocol, please refer to Zhou et al.1.

Keywords: Cell Biology; Cell culture; Cell isolation; Flow Cytometry; Genetics; Molecular Biology.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Representative FACS profiles generated from this protocol All the channels must be in logarithmic view. For SG gating, display all events in a graph [y-axis = mCherry; x-axis = SSC-A]. Draw gate around the population of SGs which have a higher mCherry signal intensity as shown in middle panel. Red indicates gated SGs and blue indicates all other particles in the cell lysates.
Figure 2
Figure 2
Representative images generated from this protocol 4sU+UVA-induced SGs in cells (left panel) and isolated by FANCI (right panel).] White dash line marks the SG-containing cells. Scale bars, 5 μm.
See this image and copyright information in PMC

References

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    1. Khong A., Matheny T., Jain S., Mitchell S.F., Wheeler J.R., Parker R. The Stress Granule Transcriptome Reveals Principles of mRNA Accumulation in Stress Granules. Mol. Cell. 2017;68:808–820.e5. doi: 10.1016/j.molcel.2017.10.015. - DOI - PMC - PubMed

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