Name: immunofluorescence analysis of stress granule formation after bacterial challenge of mammalian cells
Uploaded: 2017-07-03T13:00:00
Description: Watch this Scientific Journal Video about Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells at JoVE.com

PS is a recipient of the Bill and Melinda Gates Grand Challenge Grant OPP1141322. PV was supported by a Swiss National Science Foundation Early Postdoc Mobility fellowship and a Roux-Cantarini postdoctoral fellowship. PJS is supported by an HHMI grant and ERC-2013-ADG 339579-Decrypt.

1. Preparation of Bacteria and Host Cells
<ol>
	<li>Transfer 12 or 14 mm glass cover slips in either 24- or 12-well plates, respectively, with sterile tweezers, counting one well per experimental condition. Sterilize these by UV treatment in a tissue culture hood for 15 min. 
	NOTE: Include control wells for bacterial challenge and for SG induction by exogenous stresses.</li>
	<li>For a 24-well plate with 12 mm coverslips, seed 1 x 105 mammalian cells (e.g., HeLa cells) onto coverslips; press t...

<ol>
	<li>Reineke, L. C., Lloyd, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversion+of+stress+granules+and+P-bodies+during+viral+infection.">Diversion of stress granules and P-bodies during viral infection.</a> Virology. 436 (2), 255-267 (2013).</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>Vonaesch, P., Campbell-Valois, F. -. X., Dufour, A., Sansonetti, P. J., Schnupf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shigella+flexneri+modulates+stress+granule+composition+and+inhibits+stress+granule+aggregation.">Shigella flexneri modulates stress granule composition and inhibits stress granule aggregation.</a> Cell Microbiol. 18 (7), 982-997 (2016).</li><li>Kolobova, E., Efimov, A., 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=Microtubule-dependent+association+of+AKAP350A+and+CCAR1+with+RNA+stress+granules.">Microtubule-dependent association of AKAP350A and CCAR1 with RNA stress granules.</a> Exp Cell Res. 315 (3), 542-555 (2009).</li><li>Anderson, P., Kedersha, N. <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:+the+Tao+of+RNA+triage.">Stress granules: the Tao of RNA triage.</a> Trends in biochemical sciences. 33 (3), 141-150 (2008).</li><li>Anderson, P., Kedersha, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stressful+initiations.">Stressful initiations.</a> J Cell Sci. 115, 3227-3234 (2002).</li><li>Mohr, I., 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=Host+translation+at+the+nexus+of+infection+and+immunity.">Host translation at the nexus of infection and immunity.</a> Cell Host Microbe. 12 (4), 470-483 (2012).</li><li>Hu, S., Claud, E. C., Musch, M. W., Chang, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granule+formation+mediates+the+inhibition+of+colonic+Hsp70+translation+by+interferon-+and+tumor+necrosis+factor.">Stress granule formation mediates the inhibition of colonic Hsp70 translation by interferon- and tumor necrosis factor.</a> Am J Physiol Gastointest Liver Physiol. 298 (4), 481-492 (2010).</li><li>Barry, E. M., Pasetti, M. F., Sztein, M. B., Fasano, A., Kotloff, K. L., Levine, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+pitfalls+in+Shigella+vaccine+research.">Progress and pitfalls in Shigella vaccine research.</a> Nat Rev Gastroenterol Hepatol. 10 (4), 245-255 (2013).</li><li>Lanata, C. F., Fischer-Walker, C. L., 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=Global+Causes+of+Diarrheal+Disease+Mortality+in+Children.">Global Causes of Diarrheal Disease Mortality in Children.</a> PloS One. 8 (9), 72788 (2013).</li><li>Ashida, H., Ogawa, M., 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=Shigella+deploy+multiple+countermeasures+against+host+innate+immune+responses.">Shigella deploy multiple countermeasures against host innate immune responses.</a> Curr Opin Microbiol. 14 (1), 16-23 (2011).</li><li>Ashida, H., Ogawa, M., Mimuro, H., Kobayashi, T., Sanada, T., Sasakawa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shigella+are+versatile+mucosal+pathogens+that+circumvent+the+host+innate+immune+system.">Shigella are versatile mucosal pathogens that circumvent the host innate immune system.</a> Curr Opin Immunol. 23 (4), 448-455 (2011).</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>Ray, K., Marteyn, B., Sansonetti, P. J., Tang, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+on+the+inside:+the+intracellular+lifestyle+of+cytosolic+bacteria.">Life on the inside: the intracellular lifestyle of cytosolic bacteria.</a> Nat Re. Micro. 7 (5), 333-340 (2009).</li></ol>

The protocol outlined here describes the induction, localization, and analysis of SGs in non-infected cells and cells infected with the cytosolic pathogen S. flexneri in the presence or absence of exogenous stress. Using free imaging software, the protocols allows for the precise qualitative and quantitative analysis of SG formation to identify and statistically address differences in given phenotypes.
There are several critical steps within the protocol for the infection, SG-inductio...

Visualizing host-pathogen interactions on a cellular level is a powerful method for gaining insights into pathogenic strategies and for identifying key cellular pathways. Indeed, pathogens can be used as tools to pinpoint important cellular targets or structures, as pathogens have evolved to subvert central cellular processes as a strategy for their own survival or propagation. Visualization of cellular components can be achieved by recombinantly expressing fluorescently-tagged host proteins. While this allows for real-time analysis, the generation of cell lines with specifically-tagged host proteins is highly laborious and may result in undesirable side effects. More convenient is the detection of cellular factors using specific antibodies, because multiple host factors can be analyzed simultaneously and one is not limited to a particular cell type. A drawback is that only a static view can be captured as immunofluorescence analysis necessitates host cell fixation. However, an important advantage of immunofluorescence imaging is that it readily lends itself to both qualitative and quantitative analysis. This in turn can be used to obtain statistically significant differences to provide new insights into host-pathogen interactions.
Fluorescent image analysis programs are powerful analytical tools for performing 3D and 4D analysis. However, the high cost of software and its maintenance make methods based on free open source software more widely attractive. Careful image analysis using bio-analysis software is valuable as it substantiates visual analysis and, when assigning statistical significances, increases confidence in the correctness of a given phenotype. Previously, SGs have been analyzed using the free ImageJ software, which necessitates the manual identification of individual SGs4. Here we provide a protocol for the induction and analysis of cellular SG formation in the context of bacterial infections using the free open source bio-image analysis software ICY (http://icy.bioimageanalysis.org). The bio-image analysis software has a built-in spot detector program that is highly suitable for SG analysis. It allows the fine-tuning of the automated detection process in specified Regions Of Interest (ROIs). This overcomes the need for manual analysis of individual SGs and removes sampling bias.
Many environmental stresses induce the formation of SGs, which are phase dense cytosolic, non-membranous structures of 0.2 - 5 &#181;m in diameter5,6. This cellular response is evolutionarily conserved in yeast, plants and mammals and occurs when global protein translation is inhibited. It involves aggregation of stalled translation initiation complexes into SGs, which are considered holding places for translationally-inactive mRNAs, allowing selective translation of a subset of cellular mRNAs. Upon removal of the stress, SGs dissolve and global rates of protein synthesis resume. SGs are composed of translation elongation initiation factors, proteins involved in RNA metabolism, RNA-binding proteins, as well as scaffolding proteins and factors involved in host cell signaling2, although the exact composition can vary depending on the stress applied. Environmental factors that induce SG formation include amino acid starvation, UV irradiation, heat shock, osmotic shock, endoplasmic reticulum stress, hypoxia and viral infection2,7,8. Much progress has been made in understanding how viruses induce and also subvert SG formation, while little is still known about how other pathogens, such as bacterial, fungal or protozoan pathogens, affect this cellular stress response1,7.
Shigella flexneri is a gram-negative facultative cytosolic pathogen of humans and the causative agent of severe diarrhea or shigellosis. Shigellosis is a major public health burden and leads to 28,000 deaths annually in children under 5 years of age9,10. S. flexneri infects the colonic epithelium and spreads cell-to-cell by hijacking the host&#39;s cytoskeletal components11,12. Infection of the epithelium supports the replication of S. flexneri within the cytosol but infected macrophages die through an inflammatory cell death process called pyroptosis. Infection leads to a massive recruitment of neutrophils and severe inflammation that is accompanied by heat, oxidative stress and tissue destruction. Thus, while infected cells are subject to internal stresses induced by infection, such as Golgi disruption, genotoxic stress and cytoskeletal rearrangements, infected cells are also subjected to environmental stresses due to the inflammatory process.
Characterization of the effect of S. flexneri infection on the ability of cells to respond to environmental stresses using a number of SG markers has demonstrated that infection leads to qualitative and quantitative differences in SG composition3. However, little is known about other bacterial pathogens. Here we describe a methodology for the infection of host cells with the cytosolic pathogen S. flexneri, the stressing of cells with different environmental stresses, the labeling of SG components, and the qualitative and quantitative analysis of SG formation and composition in the context of infected and non-infected cells. This method is widely applicable to other bacterial pathogens. In addition, the image analysis of the SG formation may be used for infections by viruses or other pathogens. It can be used to analyze SG formation upon infection or the effect of infection on SG formation in response to exogenous stresses.

<table border="0" cellpadding="0" cellspacing="0" width="1191">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Primary Antibodies</td>
			<td style="width:211px;"></td>
			<td style="width:76px;"></td>
			<td style="width:672px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF3b (N20), origin goat </td>
			<td>Santa Cruz</td>
			<td>sc-16377</td>
			<td>Robust and widely used SG marker. Cytosolic staining allows cell delineation. Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF3b (A20), origin goat </td>
			<td>Santa Cruz</td>
			<td>sc-16378</td>
			<td>Same target as eIF3b (N20) and in our hands was identical to eIF3b (N20). Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF3A (D51F4), origin rabbit (MC: monoclonal)</td>
			<td>Cell Signaling</td>
			<td>3411</td>
			<td>Part of multiprotein eIF3 complex with eIF3b . Dilution 1 in 800</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF4AI, origin goat </td>
			<td>Santa Cruz</td>
			<td>sc-14211</td>
			<td>Recommended by (Ref # 13). Dilution 1 in 200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF4B, origin rabbit</td>
			<td>Abcam</td>
			<td>ab186856</td>
			<td>Good stress granule marker in our hands. Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF4B, origin rabbit</td>
			<td>Cell Signaling</td>
			<td>3592</td>
			<td>Recommended by Ref # 13. Dilution 1 in 100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eIF4G, origin rabbit</td>
			<td>Santa Cruz</td>
			<td>sc-11373</td>
			<td>Widely used SG marker. (Ref # 13): may not work well in mouse cell lines. Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">G3BP1, origin rabbit (MC: monoclonal)</td>
			<td>BD Biosciences</td>
			<td>611126</td>
			<td>Widely used SG marker. Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tia-1, origin goat </td>
			<td>Santa Cruz</td>
			<td>sc-1751</td>
			<td>Widely used SG marker. Can also be found in P bodies when SG are present (Ref # 13). Dilution 1 in 300</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Alexa-conjugated Secondary Antibodies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A488 anti-goat , origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A-11055</td>
			<td>Cross absorbed. Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A568 anti-goat, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A-11057</td>
			<td>Cross absorbed. Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A488 anti-mouse, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A-21202</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A568 anti-mouse, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A10037</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A647 anti-mouse, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A31571</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A488 anti-rabbit, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A-21206</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A568 anti-rabbit, origin donkey</td>
			<td>Thermo Fisher</td>
			<td>A10042</td>
			<td>Dilution 1 in 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Other Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shigella flexneri</td>
			<td></td>
			<td></td>
			<td>Available from various laboratories by request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone Casein Soya (TCS) broth</td>
			<td>BD Biosciences</td>
			<td>211825</td>
			<td>Standard growth medium for Shigella, application - bacterial growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TCS agar</td>
			<td>BD Biosciences</td>
			<td>236950</td>
			<td>Standard growth agar for Shigella, application - bacterial growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Congo red</td>
			<td>SERVA Electrophoresis GmbH</td>
			<td>27215.01</td>
			<td>Distrimination tool for Shigell that have lost the virulence plasmid, application - bacterial growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly L lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P1274</td>
			<td>Useful to coating bacteria to increase infection, application - infection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G1397</td>
			<td>Selective killing of extracellular but not cytosolic bacteria, application - infection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Life Technologies</td>
			<td>15630-056</td>
			<td>PH buffer useful when cells are incubated at room-temperature, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Life Technologies</td>
			<td>31885</td>
			<td>Standard culture medium for HeLa cells, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal calf serum</td>
			<td>Biowest</td>
			<td>S1810-100</td>
			<td>5% supplementation used for HeLa cell culture medium, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-essential amino acids</td>
			<td>Life Technologies</td>
			<td>11140</td>
			<td>1/100 dilution used for HeLa cell culture medium, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td>Reagent diluent, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium arsenite</td>
			<td>Sigma-Aldrich</td>
			<td>S7400</td>
			<td>Potent stress granule inducer (Note: highly toxic, special handling and disposal required), application - stress inducer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clotrimazole</td>
			<td>Sigma-Aldrich</td>
			<td>C6019</td>
			<td>Potent stress granule inducer (Note:health hazard, special handling and disposal required), application - stress inducer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Electron Microscopy Scences</td>
			<td>15714</td>
			<td>4% PFA is used for standard fixation of cells, application - fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Used at 0.03% for permeabilizationof host cells before immunofluorescent staining, application - permeabilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A647-phalloidin</td>
			<td>Thermo Fisher</td>
			<td>A22287</td>
			<td>Dilution is at 1/40, best added during 2ary antibody staining, application - staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>Nucleid acid stain used to visualize both the host nucleus and bacteria, application - staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Parafilm</td>
			<td>Sigma-Aldrich</td>
			<td>BR701501</td>
			<td>Paraffin film useful for immunofluorescent staining of coverslips, application - staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolong Gold</td>
			<td>Thermo Fisher</td>
			<td>36930</td>
			<td>Robust mounting medium that works well for most fluorophores , application - mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mowiol</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td>Cheap and robust mounting medium that works well for most fluorophores, application - mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well cell culture plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3527</td>
			<td>Standard tissue culture plates, application - cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-mm glass coverslips</td>
			<td>NeuVitro</td>
			<td>1001/12</td>
			<td>Cell culture support for immunofluorescent applications, application - cell support</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">forceps</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td>Cheap and obust mounting medium that works well for most fluorophores, application - mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Programs and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism</td>
			<td>GraphPad Software</td>
			<td></td>
			<td>Data analysisand graphing program with robust statistical test options, application - data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica SP5</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Confocal microsope, application - image acquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td>Professional image analysis program, application - data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>Data analysis and graphing program, application - data analysis</td>
		</tr>
	</tbody>
</table>

immunofluorescence analysis of stress granule formation after bacterial challenge of mammalian cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

To explain and demonstrate the protocol described in this manuscript, we characterized the image of clotrimazole-induced SGs in HeLa cells infected or not with the cytosolic pathogen S. flexneri. An outline of the procedure is presented in Figure 1, and includes virulent and avirulent S. flexneri streaked on Congo Red plates, bacteria preparation, infection, addition of environmental stress, sample fixation and staining, sample imaging and q...

Watch this Scientific Journal Video about Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells at JoVE.com

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

The overall goal of this experiment is to assess the effects of a bacterial infection on the ability of host cells to form stress granules to response to exogenous stress. This method is a valuable tool in the field of cellular microbiology for assessing whether or not a given pathogen is able to alter or subvert the whole stress granule pathway. The main advantage of this technique is that stress granules can be qualitatively and quantitatively be analyzed in a rigorous and automated manner using free image analysis programs.This methods provides insight into the dynamics of stress granule formation and composition and how these parameters are affected by a specific pathogen. Thank you. Before beginning the challenge, inoculate a red S.flexneri M90T bacteria colony from a freshly streaked tryptic soy agar 1%Congo red agar plate into a 15 milliliter conical tube, containing eight milliliters of tryptic soy broth.After tightening the lid, incubate the colony overnight with shaking at 222 RPM and 30 degrees Celsius. The next day, subculture 150 microliters of the bacteria in eight milliliters of fresh tryptic soy broth for about two hours at 37 degrees Celsius and 222 RPM to initiate a late exponential phase induction of virulence gene expression and to enhance the bacterial infectability. When the optical density of the subculture is between 0.6 and 0.9, transfer one milliliter of the bacteria into a 1.5 milliliter tube for their collection by centrifugation.And re-suspend the pellet in infection medium at an optical density of one. Next, aspirate the supernatants from each well of a HeLa cell coverslip culture. And carefully wash the HeLa cells with 500 microliters of fresh room temperature HeLa cell medium per well.Replace the wash with 500 microliters of infection medium per well, and centrifuge the 24 well plate to spin the bacteria onto the cells. Transfer the settled bacterial cocultures into a 37 degree Celsius tissue culture incubator for 30 minutes to facilitate the host cell infection. At the end of the incubation, remove the exogenous bacteria from the cells with three rinses in one milliliter of fresh 37 degree Celsius HeLa culture medium per wash.Then cover the infected cells with one milliliter of fresh culture medium containing gentamicin and return the plate to the cell culture incubator. At the end of the incubation, replace the medium with 500 microliters of the appropriate culture medium, supplemented with the stressor of interest, and return the cells to the tissue culture incubator. After one hour, aspirate the supernatant completely, and fix the samples in 0.5 milliliters of room temperature 4%paraformaldehyde in PBS for 30 minutes.Next, discard the fixative into the appropriate waste container, and wash the coverslips with one milliliter of tris-buffered saline for two minutes with gentle shaking. At the end of the wash, completely aspirate the TBS and permeabilize the cells in each well with 500 microliters of 0.3%Triton X-100 in TBS. After 10 minutes, replace the permeabilizing solution with one milliliter of TBS, swirl the plate gently, and replace the TBS with one milliliter of blocking solution for one hour at room temperature.To label the cells with a primary antibody cocktail of interest, spot 50 microliters of the primary antibody mix per coverslip onto a piece of plastic Parafilm firmly taped onto the bench top. Then use tweezers to pick up the first coverslip. And dab the edge of the coverslip onto a piece of tissue paper to remove the excess liquid.Carefully place the coverslip onto the drop and incubate the coverslips under the appropriate labeling conditions. At the end of the primary antibody labeling incubation, transfer each coverslip into an individual well of a new 24 well plate containing one milliliter of TBS per well, and wash the cells at room temperature on a plate shaker for five minutes three times. After the last wash, label the cells on each coverslip with the appropriate secondary antibody cocktail mix, as just demonstrated.And incubate the coverslips in the dark for one hour at room temperature. At the end of the secondary antibody labeling incubation, wash the cells in one milliliter of PBS three times in a new 24 well plate with gentle shaking as just demonstrated, but protected from light. After the last wash, briefly dip each coverslip in deionized water to remove the salt, dab the coverslip edges onto a tissue, and carefully mount the coverslips onto five microliters of fluorescence mounting medium per glass microscope slide without bubbles.Then seal the coverslip with nail polish. And store the slides as appropriate until imaging. To image the cells by fluorescence confocal microscopy, begin by selecting the appropriate objective and the appropriate fluorescent channels for image acquisition.Then acquire image stacks of the cells, using the appropriate imaging software. When all of the images have been captured, use the appropriate imaging analysis software to collapse the image stacks and save the stacks as TIFF files. Next, load a control and an experimental TIFF image to the image analysis platform, and select Lookup Table to open the channel parameters in the Sequence window.Click on all of the channel tab checkboxes to deactivate all of the channels. Then click channel zero, and click on the channel zero color bar to select the preferred color, increasing the intensity of the channel as necessary to visualize all of the structures. After selecting and adjusting each of the appropriate color channels for the experimental analysis, select the canvas tab and adjust the zoom tab to visualize about 10 cells per viewing field.Using the polygon tool, click on the cell of interest and extend the line around the cell to delineate the cell boundary. To name this region of interest, in the region of interest window, right-click on the cell of interest, and double-click on the region of interest's name to modify it. After selecting and naming all of the cells of interest in the same manner, open the Spot Detector application within the detection and tracking tab.Open the Pre Processing tab and select the channel to be analyzed. Within the Detector tab, select Detect bright spots over dark background and select the appropriate scale and sensitivity. Under the Region of Interest tab, select the suggested region of interest from the sequence.Under the Filtering tab, select NoFiltering. In the Output tab, select the appropriate output. Select Remove previous spots rendered as regions of interest and click no file selected to select the folder for saving the file.Then under the Display tab, select the appropriate options of interest and click Start Detection. Within the imaging analysis software, the spot detector can be used to identify the stress granules within each of the designated regions of interest. A binary image can then be generated to display all of the identified stress granules.Stress granule analysis must be carefully tailored to each stress granule market analyzed. For example, changing the size requirement of the region of interest detected, or changing the sensitivity of the detection parameters lead to varying results. At higher pixel size scales, smaller stress granules will not be counted, and the numbers of stress granules will decrease.While increasing the sensitivity results in an increase in the number of stress granules. For example, in this experiment, a scale of two with a sensitivity of 100 gave the best result from the stress granule marker G3BP1. While a scale of two with a sensitivity of 55 gave the best result for the EIF3B stress granule marker.The surface area can then be calculated from the size of the stress granules. Frequency distribution plots are useful for highlighting shifts in the size of stress granules between different cell populations. In addition, the intensity of the fluorescence can provide information about the quality of the stress granules analyzed.Once mastered, the whole cell challenge can be done in about six to seven hours, and the analysis can be completed in another two to three hours. While attempting this procedure, it's important to remember to always process the control and the experimental samples at the same time and under the same conditions to minimize variability within the data. Following this procedure, the analysis can also be extended to a three-dimensional volume to get additional insights into stress granule localization.This semi-automated procedure allows for a rigorous and unbiased analysis of stress granules in uninfected and infected cells. It can reveal previously unknown subversions of the stress granule pathway. After watching this video, you should have a good understanding of how to infect cells with bacteria, how to induce stress granule formation, and how to use a semi-automated approach for analyzing stress granules in a qualitative and quantitative manner.And don't forget that with Shigella flexneri and stress granule inducing agents like clotrimazole can be hazardous, and that precautions such as wearing gloves, working in a BL2 lab, and properly discarding all of the reagents are necessary.

Research

JoVE Journal

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