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
<ol>
	<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...

<ol>
	<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. D., 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=Mechanistic+insights+into+mammalian+stress+granule+dynamics.">Mechanistic insights into mammalian stress granule dynamics.</a> J Cell Biol. 215 (3), 313-323 (2016).</li><li>Anderson, P., Kedersha, N., Ivanov, 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,+P-bodies+and+cancer.">Stress granules, P-bodies and cancer.</a> Biochim Biophys Acta. 1849 (7), 861-870 (2015).</li><li>Aulas, A., Van de Velde, ., C, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+stress+granule+dynamics+driven+by+TDP-43+and+FUS:+a+link+to+pathological+inclusions+in+ALS?.">Alterations in stress granule dynamics driven by TDP-43 and FUS: a link to pathological inclusions in ALS?.</a> Front Cell Neurosci. 9, 423 (2015).</li><li>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=Post-transcriptional+regulatory+networks+in+immunity.">Post-transcriptional regulatory networks in immunity.</a> Immunol Rev. 253 (1), 253-272 (2013).</li><li>Wolozin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+protein+aggregation+run+amuck:+stress+granules+and+the+genesis+of+neurodegenerative+disease.">Physiological protein aggregation run amuck: stress granules and the genesis of neurodegenerative disease.</a> Discov Med. 17 (91), 47-52 (2014).</li><li>Lloyd, R. 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P., 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=YB-1+regulates+stress+granule+formation+and+tumor+progression+by+translationally+activating+G3BP1.">YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1.</a> J Cell Biol. 208 (7), 913-929 (2015).</li><li>Adjibade, P., 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=Sorafenib,+a+multikinase+inhibitor,+induces+formation+of+stress+granules+in+hepatocarcinoma+cells.">Sorafenib, a multikinase inhibitor, induces formation of stress granules in hepatocarcinoma cells.</a> Oncotarget. , (2015).</li><li>Fournier, 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 ...

The overall goal of this workflow is to determine whether foci identified within the cytoplasm are bona fide stress granules which are RNA granules that play important roles in cell physiology. The main advantage of this workflow is that it tests for all the fundamental properties of stress granules. Though this workflow is presented for human osteosarcoma U-2 OS cells, it can also be applied to other types of cell lines and primary cells.Generally, individuals new to this method will struggle because not all stress induced foci are stress granules. Our workflow comprises all experimentation necessary to distinguish them. I will be demonstrating this procedure with Anais Aulus.We are post doctoral fellows in the lab of Paul Anderson and Pavel Ivanov. To begin, seed cells onto coverslips placed in 24 well plates as described in the text protocol. After overnight incubation, aspirate the culture media from wells B1 to B4 and from C1 to C4 of the 24 well plate.Add 500 microliters of the medium supplemented with 100 micromolars sodium arsenite to wells B1 and B2, then add 500 microliters of the medium supplemented with 50 micromolar sodium arsenite to wells B3 and B4.Next, add 500 microliters of the medium containing 150 micromolar vinorelbine to wells C1 and C2.Finally, add 500 microliters of the medium supplemented with 125 micromolar of vinorelbine to wells C3 and C4.Incubate the plate for 30 minutes at 37 degrees Celsius. After 30 minutes of incubation, add to the cells in column two five microliters of one milligram per milliliter cycloheximide solution and to cells in column four, two microliters of 1.25 milligrams per milliliter puromycin. Gently shake the plate to mix the solutions and return the plate to the incubator for an additional 30 minutes of incubation.To fix the cells, remove the media from the wells and wash the cells with approximately 250 microliters of PBS. Add approximately 250 microliters of a buffered 4%paraformaldehyde solution to fix the cells, making sure the top of the coverslip is completely covered. Then, leave the plate on a bench rocker at room temperature for 15 minutes.Next, remove and properly discard the paraformaldehyde. Add approximately 250 microliters of ice cold methanol to permeabilize and flatten the cells. Incubate the plate for an additional five minutes at room temperature on a rocker.Once the permeabilization is complete, discard the methanol and block the cells by applying approximately 250 microliters of a blocking buffer for one hour at room temperature. Then, prepare the primary antibody solution by adding 12 microliters of the antibodies against G3BP1 eIF4G and eIF3B to three milliliters of blocking buffer. Add 250 microliters of the antibody solution to each well of the 24 well plate.Incubate the plate on a rocker for at least one hour at room temperature. After one hour of incubation, remove the antibody solution and wash the cells with approximately 250 microliters of PBS for five minutes. Next, add 12 microliters of Anti-Mouse Cy2, Anti-Rabbit Cy3 and Anti-Goat Cy5 antibodies together with three microliters of hooks dye to three milliliters of blocking buffer to prepare the secondary antibody solution.Add 250 microliters of the secondary antibody solution to each well and cover the plate to protect the samples from light. Incubate the plate on a rocker at room temperature for one hour. When the incubation is completed, remove the antibody solution and wash the cells with PBS for five minutes.Heat mounting medium in a 37 degree Celsius heat block for 10 minutes to reduce viscosity. Then using a precut 200 microliter pipette tip, place 25 microliters of the mounting medium onto a labeled glass slide. With fine forceps, transfer the coverslip from the well to the slide, inverting the top so that the cells face the mounting medium.Use a clean P200 tip to press the coverslip down. Once all the coverslips are mounted, remove the excess mounting medium by pressing a lab tissue firmly against the slide. Afterwards, flush the slide with water.And immediately blot it with lab tissue once again. Permeabilize the prefixed cells by incubating them with 250 microliters of ice cold methanol at room temperature for 10 minutes. Then, to the emptied wells of the plate add approximately 250 microliters of 70%ethanol and seal the plate using parafilm to prevent evaporation.Incubate the plate at four degrees Celsius overnight. After the overnight incubation, remove the ethanol and rehydrate the cells by adding 500 microliters of 2XSSC. Incubate the coverslips for five minutes on a rocker at room temperature.Then, discard the solution and add 500 microliters of SSC once more. Incubate the plate for an additional five minutes with gentle agitation. Next, layer the parafilm on the bottom of a hybridization oven and pipette 25 microliters of a hybridization buffer on top of it.Transfer the coverslip from the 24 well plate, inverting the top so that the cells face the hybridization buffer. Incubate the coverslips at 42 degrees Celsius for 15 minutes. Pipette 25 microliters of a biotin oligo dT solution onto the parafilm in the hybridization oven.Then, using forceps, pick up the coverslip and blot its edge against a lab tissue to remove excess hybridization buffer. Transfer the coverslip to the biotin oligo dT solution, making sure the cells are face down and incubate at 42 degrees Celsius for 60 minutes. Once the hybridization is completed, add approximately 500 microliters of prewarmed 2XSSC to the wells of the 24 well dish.Then, using forceps, transfer the coverslip to the well and incubate it at 42 degrees Celsius for 10 minutes. After further staining the cells with antibody solution, view the specimens using a fluorescence microscope. Presented here are images of the untreated U-2 OS cells that do not contain any granules or foci stained with stress granule markers G3BP1, eIF4G, and eIF3B.In U-2 OS cells treated with sodium arsenite or vinorelbine, the formation of cytoplasmic foci that contain G3BP1, eIF4G and eIF3B was induced. The co-localization of these markers was confirmed by line scan analysis performed for each channel at increased magnification followed by super imposition of the obtained graphs. Additionally, cytoplasmic foci identified in the treated cells contained mRNA as shown by poly A FISH.The oligo dT signal co-localized with the G3BP1 signal confirming the identify of stress granules. Once mastered, this entire workflow can be done in three days if it's performed properly. After watching this video, you should have a good understanding of how to perform immunofluorescence and fluorescent hybridization.Steps that are critical for the correct characterization and identification of stress granules. Don't forget that working with paraformaldehyde could be extremely hazardous. Precautions such as use of PPE and adequate ventilation should always be used while performing this procedure.

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14.4K vistas.  Brigham and Women's Hospital. The overall goal of this workflow is to determine whether foci identified within the cytoplasm are bona fide stress granules which are RNA granules that play important roles in cell physiology. The main advantage of this workflow is that it tests for all the fundamental properties of stress granules. Though this workflow is presented for human osteosarcoma U-2 OS cells, it can also be applied to other types of cell lines and primary cells.Generally, individuals new to this method will ...