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 ...

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.

methods-to-classify-cytoplasmic-foci-as-mammalian-stress-granules

Research

JoVE Journal

Biology

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice&#160;and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer&#39;s disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.

Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

Traditionally used for bulk biochemical assays, Xenopus laevis egg extracts have emerged as a powerful imaging-based tool for studying cytoplasmic phenomena, such as cytokinesis, mitotic spindle formation and assembly of the nucleus. Building upon early methods that imaged fixed extracts sampled at sparse time points, recent approaches image live extracts using time-lapse microscopy, revealing more dynamical features with enhanced temporal resolution. These methods usually require sophisticated surface treatments of the imaging vessel. Here we introduce an alternative method for live imaging of egg extracts that require no chemical surface treatment. It is simple to implement and utilizes mass-produced laboratory consumables for imaging. We describe a system that can be used for both wide-field and confocal microscopy. It is designed for imaging extracts in a 2-dimensional (2D) field, but can be easily extended to imaging in 3D. It is well-suited for studying spatial pattern formation within the cytoplasm. With representative data, we demonstrate the typical dynamic organization of microtubules, nuclei and mitochondria in interphase extracts prepared using this method. These image data can provide quantitative information on cytoplasmic dynamics and spatial organization.

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization

We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs.