Name: methods to classify cytoplasmic foci as mammalian stress granules
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Description: Watch this Scientific Journal Video about Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules at JoVE.com

We thank members of the Ivanov and Anderson labs for their helpful discussion and feedback on this manuscript. This work was supported by the National Institutes of Health [GM111700 and CA168872 to PA, NS094918 to PI], the National Science Centre in Poland [grant UMO-2012/06/M/NZ3/00054 to WS]. WS also acknowledges the Ministry of Science and Higher Education in Poland (Mobility Plus Program) and the Polish-American Fulbright Commission for their financial support of his research in the USA.

1. Cell Preparation
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	<li>Add autoclaved coverslips to 12 wells of a 24-well plate, as indicated in Figure 1A; this can be done using a sterile Pasteur pipette attached to a vacuum to pick up each coverslip. Gently tap the coverslip on the side of the well to release the suction, allowing the coverslip to fall into the well.</li>
	<li>Plate 1 x 105 U-2 OS (U2OS) osteosarcoma cells per well at a final volume of 500 &#181;L of medium. Move the plate side to side and up and down several tim...

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	<li>Holcik, M., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+in+stress+and+apoptosis.">Translational control in stress and apoptosis.</a> Nat Rev Mol Cell Biol. 6 (4), 318-327 (2005).</li><li>Yamasaki, S., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+mRNA+translation+during+stress.">Reprogramming mRNA translation during stress.</a> Curr Opin Cell Biol. 20 (2), 222-226 (2008).</li><li>Buchan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNP+granules:+Assembly,+function,+and+connections+with+disease.">mRNP granules: Assembly, function, and connections with disease.</a> RNA Biol. 11 (8), (2014).</li><li>Kedersha, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+ternary+complex+(eIF2-GTP-tRNA(i)(Met))-deficient+preinitiation+complexes+are+core+constituents+of+mammalian+stress+granules.">Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules.</a> Mol Biol Cell. 13 (1), 195-210 (2002).</li><li>Kedersha, N., Ivanov, P., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granules+and+cell+signaling:+more+than+just+a+passing+phase?.">Stress granules and cell signaling: more than just a passing phase?.</a> Trends Biochem Sci. 38 (10), 494-506 (2013).</li><li>Kedersha, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+shuttling+of+TIA-1+accompanies+the+recruitment+of+mRNA+to+mammalian+stress+granules.">Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules.</a> J Cell Biol. 151 (6), 1257-1268 (2000).</li><li>Bley, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granules+are+dispensable+for+mRNA+stabilization+during+cellular+stress.">Stress granules are dispensable for mRNA stabilization during cellular stress.</a> Nucleic Acids Res. 43 (4), e26 (2015).</li><li>Kedersha, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G3BP-Caprin1-USP10+complexes+mediate+stress+granule+condensation+and+associate+with+40S+subunits.">G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits.</a> J Cell Biol. 212 (7), 845-860 (2016).</li><li>Panas, M. 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L., Gupta, M., Li, W., Miller, I., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-binding+proteins+TIA-1+and+TIAR+link+the+phosphorylation+of+eIF-2+alpha+to+the+assembly+of+mammalian+stress+granules.">RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules.</a> J Cell Biol. 147 (7), 1431-1442 (1999).</li><li>Kedersha, N., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+stress+granules+and+processing+bodies.">Mammalian stress granules and processing bodies.</a> Methods Enzymol. 431, 61-81 (2007).</li><li>Kedersha, N., Tisdale, S., Hickman, T., Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+and+quantitative+imaging+of+mammalian+stress+granules+and+processing+bodies.">Real-time and quantitative imaging of mammalian stress granules and processing bodies.</a> Methods Enzymol. 448, 521-552 (2008).</li><li>Langer-Safer, P. 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As evidenced by immunohistological studies in multiple diseases, chronic stress leads to the formation of different intracellular foci. For example, most neurodegenerative diseases are characterized by intracellular aggregates of insoluble proteins. The presence of SG-associated proteins in such aggregates is often the basis for concluding that such foci are SGs. A similar conclusion is also drawn when SG marker-positive cytoplasmic foci are observed in cells treated with new stress stimuli. This protocol provides a simp...

Cells employ many mechanisms to respond to stress. Some responses occur at the post-transcriptional level and involve regulating mRNA translation and/or stability1,2. Stress-modulated mRNA translational arrest and degradation are associated with the formation of specific nonmembranous cellular foci, the most well characterized being Stress Granules (SGs)3. SGs are cytoplasmic foci that concentrate nontranslating mRNPs in translationally-arrested cells responding to stress (e.g., oxidation, heat shock, nutrient starvation, and viral infection)4. In addition to nontranslating mRNAs, SGs contain translation initiation factors, RNA-binding proteins, and various signaling proteins5. SGs are biomarkers of inhibited protein translation and altered RNA metabolism and have been linked to cell survival and apoptosis, signaling pathways, and nuclear processes5.
SGs are dynamic entities, and their formation is tightly connected to the status of cellular translation6. Despite their seemingly solid appearance, most SG protein components rapidly shuttle in and out, with a residence time of seconds. While SGs persist for minutes to hours, most of their components are in rapid flux. The inhibition of translation initiation and the consequent disassembly of translating polysomes promote the formation of SGs; thus, SGs are in equilibrium with translating polysomes. This polysome/SG equilibrium is key to distinguishing bona fide SGs from other stress-induced foci6,7.
Arresting translation initiation entails the conversion of translation-competent pre-initiation complexes that contain mRNA, translation initiation factors (eIF), and 40S ribosomal subunits (so-called 48S complexes) into translationally stalled complexes (so-called 48S* complexes) that can coalesce into SGs4,6. SGs are promoted by translational arrest at two different steps upstream of 48S complex formation: interference with the functions of the cap-binding eIF4F complex (e.g., targeting its eIF4A subunit) or phosphorylation of the &#945; subunit of translation initiation factor eIF2, mediated by one or more of the four eIF2 kinases. The presence of stalled 48S complexes (i.e.&#160;40S ribosomal subunits and selected translation initiation factors) is a hallmark of SGs8,9.
SGs have been linked to various pathological states, such as viral infections, neurodegeneration, autoimmunity, and cancer3,10,11,12,13. Mutated forms of several SG components are associated with neurodegenerative diseases (e.g., amyotrophic lateral sclerosis) in which neurons display intracellular pathological inclusions that may play active roles in neuronal death13. Some viruses hijack SG components to inhibit SG formation and to enhance viral replication14. Recent studies also link SGs to cancer15 and to cancer cell survival under chemo- and radiotherapy treatments10,16,17,18,19,20. While such findings have stimulated great interest in SG biology, many of the published reports lack important controls to distinguish the formation of bona fide SGs from other stress-induced foci.
SGs were initially described as nonmembranous cytoplasmic foci composed of selected mRNA-binding Proteins (RBPs), including T-cell ntracellular antigen 1 (TIA-1) and Poly-A-binding Protein (PABP); translation initiation factors; polyadenylated mRNA; and small ribosomal subunits4,6,21,22. Traditionally, their composition has been determined by techniques such as immunostaining and the ectopic expression of fluorescently tagged proteins23,24. Due to their highly dynamic nature, the immunolocalization of SG proteins and/or mRNAs remains the defining methodology for detecting SGs23,24. To date, many RBPs and other proteins (over 120 distinct proteins) are described as SG components11. As many SG-localized proteins change their localization in both a stress-dependent and an independent manner and can accumulate at other intracellular compartments and foci, it is important to choose correct SG markers and to employ functional criteria to distinguish SGs from other types of stress-induced foci. Bona fide SGs contain mRNA, translation initiation factors, and small ribosomal subunits and are in dynamic equilibrium with translation.
This simple workflow is designed to determine whether stress-induced foci are bona fide SGs. This workflow includes several experimental approaches using U2OS cells, cells commonly used to study SGs. These cells are ideal, as they have a large cytoplasm, are relatively flat, and attach strongly to glass coverslips. Other cell types can be used to study SGs, but it is important to be aware that differences in timing, drug concentration, and abundance of SG-nucleating proteins can alter the kinetics and composition. Additionally, some cells respond to paraformaldehyde fixation by forming cell surface blebs, giving then a ruffled appearance that causes the punctate localization of some SG markers; without careful analysis, these can be misclassified as SGs. This highlights the necessity of assessing all criteria required to identify stress-induced foci as bona fide SGs. Vinorelbine (VRB), a cancer therapeutic that promotes SGs20, as well as Sodium Arsenite (SA), a robust and well-characterized SG inducer, are used as stresses for the experiments in this protocol.
Canonical SGs contain multiple SG markers (both proteins and mRNAs) that colocalize in cytoplasmic foci. This protocol uses both immunofluorescence and fluorescence in situ hybridization (FISH) to detect the localization of protein markers and polyadenylated mRNA22, respectively. Briefly, indirect immunofluorescence uses antibodies (i.e.&#160;primary antibodies) specific for a given protein to detect it within the cell. Then, secondary antibodies (usually species-specific) attached to fluorochromes recognize the primary antibody and reveal the target protein localization. Fluorescent microscopy is used to detect the localized signal within the cell. Using antibodies produced in different species and detecting them with differently colored secondary antibodies allows for the detection of the colocalization of multiple antibodies, indicating that their protein targets are found in the same location23. It is important to select markers that have been verified to colocalize at bona fide SGs.
FISH uses a labeled probe that base-pairs to a specific RNA or DNA sequence25. To detect mRNA, this protocol uses a biotinylated oligo(dT)40 probe that hybridizes (or base-pairs) to the polyA tail of mRNA (i.e.&#160;polyA FISH). The biotinylated probe is then detected using fluorescently conjugated streptavidin, as streptavidin has a high affinity for biotin. When assessing SGs, it is important to couple polyA FISH with immunofluorescence detecting an SG marker, as in bona fide SGs, the two signals should colocalize.
Using this protocol, colocalization is assessed in multiple ways. In a multi-channel (i.e.&#160;RGB: red, green, and blue) image, colocalization will alter the color of the overlapped signal (e.g.&#160;colocalized red and green appear yellow)23. Additionally, colocalization is quantified graphically using line scan analysis, where the intensity of each of color is measured across a given line8,20. This protocol describes two line scan analysis procedures using ImageJ26. One procedure is manual and goes through the entire process, while the other uses a macro, or a simple program that automates the manual steps. It is important to go through the manual program to understand the macro procedure.
SGs form in cells where translation is repressed; therefore, cells with SGs should display decreased levels of global translation compared to untreated cells. Experimentally, ribopuromycylation is used. Puromycin and emetine are added to cells for a short duration prior to fixation, allowing the puromycin to incorporate into actively forming polypeptides, causing termination27,28. Treatment with emetine is required to prevent re-initiation29. Puromycin can then be detected using anti-puromycin antibody, giving a snapshot of active translation. This method is used because it is quick, does not require pre-starving with a medium lacking a particular amino acid (and potentially prestressing the cells), and reveals the subcellular localization of protein translation. Other methods, notably using modified amino acid analogs, such as the methionine analog L-azidohomoalaine (AHA), coupled with &#34;click-it&#34; chemistry30, have been used to show that cells containing SGs exhibit much lower translation than neighboring cells31. However, this technique requires methionine starvation followed by pulse-labeling for 15-30 min. Methionine starvation constitutes an additional stress, while the long labeling time (i.e.&#160;15-30 min) results in the measurement of cumulative rather than ongoing translation and also allows the newly-synthesized proteins to move from their site of synthesis to their final destination within the cell. In contrast, ribopuromycylation is much faster and is compatible with any medium, such as the glucose-free medium used to induce SGs via glucose starvation.
Canonical SGs are in dynamic equilibrium with actively translating polysomes. This can be assessed experimentally by treating samples with the drugs that stabilize or destabilize polysomes, thus tipping the balance between SGs and polysomes23. Cycloheximide (or emetine, which behaves similarly) blocks elongation by &#34;freezing&#34; ribosomes onto mRNA, thus decreasing the pool of available non-polysomal mRNPs able to form SGs. Experimentally, this can be used in two ways: by adding cycloheximide (or emetine) before stress to prevent SG formation or by adding cycloheximide (or emetine) after the SGs have formed, even without removing the stressing agent, causing SG disassembly, as stalled preinitiation complexes are slowly admitted into the polysome fraction. In contrast, puromycin causes premature termination and promotes polysome disassembly, increasing the pool of initiating mRNAs capable of assembling into SGs. Experimentally, puromycin treatment increases the number of SGs or lowers the threshold at which they form in response to graded stress or drug dosage. When assessing the effect of puromycin, it is important to use a sub-maximal level of the drug of interest, as puromycin is expected to augment the effect of the drug and to increase the percent of cells displaying SGs &#8211; this cannot occur if the drug/stress causes SGs in 100% of the cells initially. This contrasts with cycloheximide treatment, which disassembles SGs and works best when ~95% of the cells initially display SGs so that the largest possible decrease can be observed and accurately quantified.
This protocol provides a framework to study SGs in mammalian cells. Methods include: (1) immunofluorescent staining and ImageJ analysis to assess the colocalization the classic SG-associated markers eIF4G, eIF3b, and Ras GTPase-activating protein-binding protein 1 (G3BP1) in putative SGs; (2) oligo(dT) fluorescence in situ hybridization (polyA FISH) to detect polyadenylated mRNA; (3) cycloheximide and puromycin treatment to determine whether stress-induced foci are in dynamic equilibrium with polysomes; and (4) ribopuromycylation to assess the translational status of cells containing putative SGs. Together, these assays can determine whether stress-induced foci can be classified as bona fide SGs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>U-2 OS</td>
			<td>ATCC</td>
			<td>ATCC&#174;HTB-96&#8482;</td>
			<td>- commonly written as &#34;U2OS&#34; 
			- culture at 37 &#176;C under 5% CO2 in 10% FBS/DMEM</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modification of Eagle&#39;s Medium</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td>- abbreviated DMEM 
			- contains 4.5 g/L glucose, pyruvate, and phenol red 
			- supplemented with 10% FBS, 10 mM HEPES, and penicillin/steptomycin 
			- prewarm prior drug(s) dilution</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F2442-500ml</td>
			<td>- abbreviated FBS 
			- used to supplement media</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630-080</td>
			<td>- used to supplement media</td>
		</tr>
		<tr>
			<td>penicilline streptomycine</td>
			<td>Sigma</td>
			<td>P0781-100ml</td>
			<td>- used to supplement media</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Costar</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>lab tissues</td>
			<td>KIMTECH SCIENCE</td>
			<td>34120</td>
			<td>- commonly called &#34;kimwipes&#34;</td>
		</tr>
		<tr>
			<td>Coverglass for Growth Cover Glasses</td>
			<td>Fisher Scientific</td>
			<td>12-545-82</td>
			<td>- autoclaved before used 
			- keep sterile in the tissue culture hood</td>
		</tr>
		<tr>
			<td>Sodium (meta)arsenite &#8805;90%</td>
			<td>Sigma</td>
			<td>S7400-100G</td>
			<td>- commonly called sodium arsenite and abbreviated SA 
			- dissolution in water at 1 M concentration, then dilute to 100 &#181;M as a working stock</td>
		</tr>
		<tr>
			<td>Vinorelbine</td>
			<td>TSZ CHEM</td>
			<td>RS055</td>
			<td>- abbreviated VRB 
			- dissolve in water to 10 mM</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Sigma</td>
			<td>P9620</td>
			<td>- used to assess translation level (ribopuromycylation) 
			- used to assess SG connection with active translation 
			- dilute in water to 10 mg/mL</td>
		</tr>
		<tr>
			<td>Emetine dihydrochloride hydrate</td>
			<td>Sigma</td>
			<td>E2375</td>
			<td>- used in combination with puro to assess general translation level 
			- used to assess SG connection with active translation 
			- dilute in water to 10 mg/mL</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>C4859</td>
			<td>- abbreviated CHX 
			- used to assess SG connection with active translation 
			- dilute in water to 10 mg/mL</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Lonza / VWR</td>
			<td>95042-486</td>
			<td>- abbreviated PBS 
			- wash buffer for immunofluorescence</td>
		</tr>
		<tr>
			<td>Paraformaldehyde reagent grade, crystalline</td>
			<td>Sigma</td>
			<td>P6148-500G</td>
			<td>- make a 4% solution in hot PBS in fume hood, stir until dissolved 
			- aliquots can be stored at - 20 &#176;C for several months 
			- hazardous, use ventilation 
			- discard in special waste</td>
		</tr>
		<tr>
			<td>Methanol, ACS</td>
			<td>BDH / VWR</td>
			<td>BDH1135-4LP</td>
			<td>- pre-chilled to -20&#176;C before use 
			- discard in special waste</td>
		</tr>
		<tr>
			<td>Normal Horse Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>31874</td>
			<td>- abbreviated NHS 
			- dilute to 5% in PBS 
			- add sodium azide for storage at 4&#176;C 
			- blocking solution for immunofluorescence</td>
		</tr>
		<tr>
			<td>Ethanol, Pure, 200 Proof (100%),USP, KOPTEC</td>
			<td>Decon Labs / VWR</td>
			<td>89125-188</td>
			<td>- dilute to 70% with water</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti G3BP1 antibody (TT-Y)</td>
			<td>Santa Cruz</td>
			<td>sc-81940</td>
			<td>- store at 4 &#176;C 
			- use at 1/100 dilution</td>
		</tr>
		<tr>
			<td>rabbit anti eIF4G antibody (H-300)</td>
			<td>Santa Cruz</td>
			<td>sc-11373</td>
			<td>- store at 4 &#176;C 
			- 1/250 dilution</td>
		</tr>
		<tr>
			<td>goat anti eIF3&#951; (N-20) antibody</td>
			<td>Santa Cruz</td>
			<td>sc-16377</td>
			<td>- eIF3&#951; is also known as eIF3b 
			- store at 4 &#176;C 
			- 1/250 dilution</td>
		</tr>
		<tr>
			<td>mouse anti puromycin 12D10 antibody</td>
			<td>Millipore</td>
			<td>MABE343</td>
			<td>- store at 4 &#176;C 
			- 1/1000 dilution</td>
		</tr>
		<tr>
			<td>Cy2 AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>715-225-150</td>
			<td>- reconstitute in water as per manufacturer&#8217;s instructions then store at 4&#176;C 
			- 1/250 dilution</td>
		</tr>
		<tr>
			<td>Cy3 AffiniPure Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>711-165-152</td>
			<td>- reconstitute in water as per manufacturer&#8217;s instructions then store at 4&#176;C 
			- 1/2500 dilution</td>
		</tr>
		<tr>
			<td>Cy5 AffiniPure Donkey Anti-Goat IgG (H+L)</td>
			<td>Jackson Immunoresearch</td>
			<td>705-175-147</td>
			<td>- reconstitute in water as per manufacturer&#8217;s instructions then store at 4&#176;C 
			- 1/250 dilution</td>
		</tr>
		<tr>
			<td>Hoechst 33258 solution</td>
			<td>Sigma</td>
			<td>94403-1ML</td>
			<td>- incubate with secondary antibodies 
			-stock solution 0.5 mg/mL in dH20, protect from light 
			- Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Cy3 Streptavidin</td>
			<td>Jackson Immunoresearch</td>
			<td>016-160-084</td>
			<td>- reconstitute in water as per manufacturer&#8217;s instructions, then store at 4&#176;C 
			- 1/250 dilution</td>
		</tr>
		<tr>
			<td>oligo-(dT)40x probe biotinilated</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>/</td>
			<td>- reconstitute in water to 100 ng/mL, aliquot and store at -20&#176;C 
			- dilute in hybridation buffer 1/50 prior to use 
			- custom order</td>
		</tr>
		<tr>
			<td>hybridation box</td>
			<td>/</td>
			<td>/</td>
			<td>- make a moisture chamber with any storing slide box by adding humidified paper on the bottom 
			- prewarm the box prior use</td>
		</tr>
		<tr>
			<td>20 x SSC buffer</td>
			<td>Thermo fisher Ambion</td>
			<td>AM9763</td>
			<td>- dilute to 2X SSC using RNase Free water (DEPC treated) 
			- store at room temperature 
			- wash buffer for FISH</td>
		</tr>
		<tr>
			<td>PerfectHybPlus Hybridization Buffer</td>
			<td>Sigma</td>
			<td>H7033-50ml</td>
			<td>- block and probe incubation buffer for FISH</td>
		</tr>
		<tr>
			<td>slide mounting media</td>
			<td>home made</td>
			<td>/</td>
			<td>- mix 5 g of &#8220;cold-soluble&#8221; poly(vinyl alcohol) in 20 ml of PBS 
			- mix by sonication, followed by stirring overnight at RT 
			- Add 5 ml of glycerol and 0.2 ml of 20 % sodium azide, and stir for 16 h at RT 
			- centrifugation at 20,000 g for 20 min, discard large pellet 
			- aliquot viscous liquid, long-term storage at -20&#176;C, 1 week at 4&#176;C</td>
		</tr>
		<tr>
			<td>Parafilm &#34;M&#34;</td>
			<td>Sigma</td>
			<td>P7793-1EA</td>
			<td>- usually referred as &#34;parafilm&#34;</td>
		</tr>
		<tr>
			<td>Poly(vinyl alcohol)</td>
			<td>Sigma</td>
			<td>P-8136-250G</td>
			<td>- reagent to make vinol</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516-100ML</td>
			<td>- reagent to make vinol</td>
		</tr>
		<tr>
			<td>sodium azide</td>
			<td>Fisher Scientific</td>
			<td>S2002-5G</td>
			<td>- preservative agent for blocking solution and vinol 
			- make a 20 % dilution in water</td>
		</tr>
	</tbody>
</table>

methods to classify cytoplasmic foci as mammalian stress granules

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress-induced foci are not necessarily SGs. SGs are classified as cytoplasmic foci that contain mRNAs, translation initiation factors, and RNA-binding proteins and are in equilibrium with active translation. The above protocol can be used as a template to characterize whether a given stress induces bona fide SGs.
SGs colocalize with other known SG markers, including both proteins and mRNA. SA and VRB induce cytoplasmic...

Watch this Scientific Journal Video about Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules at JoVE.com

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...

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