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

## TL;DR

This paper provides a detailed protocol for isolating stress granules in HeLa cells using a fluorescence-based method called FANCI.

## Contribution

The novel contribution is a new protocol for isolating stress granules using fluorescence-activated non-membrane condensate isolation (FANCI).

## Key findings

- A protocol is described for inducing and isolating stress granules in G3BP1-mCherry HeLa cells.
- The method uses flow cytometry for SG purification and includes RNA extraction from purified SGs.
- The protocol may be applicable to study stress granules in other cell types.

## 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

•Administering specific stress treatments to cells to induce SGs•Instructions for preparing SG-containing cell lysates•Explanation of the flow cytometry-based SG purification procedure•Procedures for RNA extraction from purified SGs

Administering specific stress treatments to cells to induce SGs

Instructions for preparing SG-containing cell lysates

Explanation of the flow cytometry-based SG purification procedure

Procedures for RNA extraction from purified SGs

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.