The work was supported by funding from the Croucher Foundation and City University startup funds. We thank all members of the Chow laboratory for help with the experiment and critical reading of the manuscript.

NOTE: The protocol described here uses CAL-1 pDC cell line treated with resiquimod (R848), an agonist for TLR7/8. This protocol has been applied to other human and murine cell types, including RAW 264.7 (murine macrophage line), THP-1 (human monocytic cell line), BJAB (human B cell line), Ramos (human B cell line), and MUTZ-3 (human dendritic cell line)23.
1. Stimulation of CAL-1 Cells
<ol>
	<li>Maintain CAL-1 cell cultures in a T75 flask at 37 &#176;C and 5% CO2 under sterile conditions with 20&#8722;25 mL of RPMI 1640 medium containing 5% fetal bovine serum (FBS), 25 mM HEPES, and 1x mercaptoethanol (i.e., complete RPMI 1640 medium).</li>
	<li>Transfer the cells into a 50 mL conical tube. 
	NOTE: CAL-1 cells are non-adherent. For adherent cell types, standard trypsinization can be performed to harvest cells.</li>
	<li>Centrifuge the cells at 200 x g for 5 min at room temperature (RT). Remove supernatant and resuspend the cell pellet in 8 mL of the complete RPMI 1640 medium to obtain a homogeneous single cell suspension.</li>
	<li>Count the cells using a hemocytometer. Seed the cells at a density of 1 x 106 cells per well in a 6 well plate with 4 mL of preheated complete RPMI 1640 medium in each well. Incubate for 20&#8722;24 h at 37 &#176;C and 5% CO2 to allow the confluency to reach 90%&#8722;95% (corresponding to approximately 1.5 x 106 cells).</li>
	<li>Stimulate the cells by adding 4 &#181;L of 1 mg/mL R848 per well of the 6 well plate (final concentration of 1 &#181;g/mL). Also set up an unstimulated control well with cells without the R848 treatment.</li>
	<li>Ensure the R848 is evenly dispersed by gently rocking the plate side to side. Then, incubate the cells for 2&#8722;16 h in the incubator at 37 &#176;C and 5% CO2.</li>
</ol>
2. Extraction of Cellular Proteins
<ol>
	<li>Transfer the cell suspensions from the 6 well plate into 5 mL centrifuge tubes.</li>
	<li>Centrifuge at 200 x g for 5 min at RT. Remove the supernatant and resuspend the cell pellet in 1 mL of phosphate-buffered saline (PBS) to obtain a homogeneous single cell suspension.</li>
	<li>Transfer the cell suspension into a 1.5 mL centrifuge tube.</li>
	<li>Spin down briefly at 12,000 x g for 0.5&#8722;1 min at 4 &#176;C and carefully remove the supernatant.</li>
	<li>Prepare the lysis buffer containing 6.25 mL of 1 M Tris-HCl pH 7.4 (final concentration of 25 mM), 7.5 mL of 5 M NaCl (final concentration of 150 mM), 0.5 mL of 0.5 M EDTA (final concentration of 1 mM), 2.5 mL of NP-40 (final concentration of 1%) and 7.5 mL of glycerol (final concentration of 5%) in 250 mL of deionized water (ddH2O). Add 100x protease inhibitor single-use cocktail to a final concentration of 1x to the lysis buffer just before use. Keep the prepared lysis buffer on ice. 
	NOTE: The lysis buffer without the protease can be stored at 4 &#176;C.</li>
	<li>Resuspend the cell pellet in 30 &#181;L of ice-cold lysis buffer and mix by pipetting up and down.</li>
	<li>Incubate on ice for 15&#8722;20 min.</li>
	<li>Clarify the lysate by centrifuging at 12,000 x g for 15&#8722;20 min at 4 &#176;C. Transfer the supernatant into a new prechilled 1.5 mL centrifuge tube. Keep the extracts on ice at all times. 
	NOTE: Cell lysates can be stored at -20 &#176;C or -80 &#176;C. Do not boil the samples.</li>
	<li>Measure protein concentration using Bradford reagent.</li>
</ol>
3. Analysis of IRF5 Dimerization by Native PAGE
<ol>
	<li>Prepare the upper (-) and lower (+) chamber electrophoresis buffers. The upper chamber buffer consists of 0.3% sodium deoxycholate (NaDOC) in 1x native PAGE running buffer, and the lower chamber consists of only 1x native PAGE running buffer. 
	NOTE: Prepare a fresh upper chamber buffer for every new run.</li>
	<li>Rinse a 3%&#8722;12% native PAGE gel thoroughly with water without distorting the wells. Set the gel into the mini gel tank and remove the comb. Prerun the gel in a 4 &#176;C cold room or on ice at 150 V for 30 min. 
	NOTE: Prerunning removes excessive ammonia and persulfate ions that can interfere with the running of the gel.</li>
	<li>During the prerun, prepare the samples for loading by mixing the cellular proteins kept on ice with 4x native sample buffer.</li>
	<li>After the prerun, load 10&#8722;15 &#181;g of protein with a final volume of 10&#8722;15 &#181;L per sample. 
	NOTE: Overloading of protein can cause smearing.</li>
	<li>Run gel at 85 V for 30 min, then 150 V for 2 h. 
	NOTE: Considering differences in equipment and cell lines used by different labs, minor modifications to the concentration of protein samples, voltage and running time may be appropriate to optimize this protocol. Lowering pre-running and running voltage while increasing running time may help to improve dimer resolution and result consistency.</li>
	<li>Soak the gel in SDS running buffer (25 mM Tris pH 8.3, 250 mM Glycine, 0.1% SDS) for 30 min at RT. 
	NOTE: No agitation is required. Occasionally, the intensity of the band may not be proportional to the amount of protein loaded due to inefficient transfer in the presence of deoxycholate (DOC), which mainly affects the monomeric form of IRF5. Soaking the gel in SDS running buffer prior to the transfer solves this problem. The gel is fragile. Handle with extreme care from the bottom (i.e., higher percentage) end of the gel.</li>
</ol>
4. Immunoblot Analysis of IRF5
<ol>
	<li>Activate the polyvinylidene difluoride (PDVF) membrane by soaking it in methanol for approximately 5 min.</li>
	<li>Make a cut on one corner of the membrane to indicate its orientation. Assemble the transfer sandwich according to the sequential order detailed in the manufacturer&#8217;s protocol with extra care to ensure that no air bubbles are trapped within.</li>
	<li>Place the transfer cassette into the tank and transfer at 20 V for 1 h on ice. 
	NOTE: Perform all incubations and washes in subsequent steps with a rocking shaker.</li>
	<li>Remove the membrane from the cassette with plastic forceps after the transfer is completed. Block the membrane in blocking buffer (TBS) for 45 min at RT. 
	NOTE: TBS buffer with 5% BSA can also be used as a blocking buffer.</li>
	<li>Incubate the membrane with the primary antibody listed in Table 1. Wash the membrane with 1x TBST washing buffer (20 mM Tris, pH 7.0, 150 mM NaCl and 0.1% Tween 20) for 3 min while rocking. Repeat the wash 2x.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Dillution</td>
			<td>Dillution buffer</td>
			<td>Incubation</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Primary antibody(Anti-IRF5)</td>
			<td>1/1,000</td>
			<td>TBS blocking buffer</td>
			<td>Overnight at 4 &#176;C or 2 h at RT</td>
			<td>Diluted antibodies can be reused several times if stored at 4 &#176;C in the presence of 0.02% sodium azide.</td>
		</tr>
		<tr>
			<td>Secondary antibody(Anti-rabbit)</td>
			<td>1/10,000</td>
			<td>TBS blocking buffer</td>
			<td>45 min at RT</td>
			<td>Diluted antibodies can be reused several times if stored at 4 &#176;C in the presence of 0.02% sodium azide.</td>
		</tr>
		<tr>
			<td colspan="5">NOTE: Dilution need to be optimized as it varies between manufacturers.</td>
		</tr>
	</tbody>
</table>
Table 1: Specifications of the antibodies used in the immunoblotting procedure.
<ol start="6">
	<li>Incubate the membrane with the secondary antibody listed in Table 1. Wash the membranes for 3 min in 1x TBST washing buffer. Repeat the wash 2x.</li>
	<li>Scan the blot using an appropriate gel documentation system.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IKKalpha+negatively+regulates+IRF-5+function+in+a+MyD88-TRAF6+pathway.">IKKalpha negatively regulates IRF-5 function in a MyD88-TRAF6 pathway.</a> Cellular Signalling. 22 (1), 117-127 (2010).</li><li>Paun, 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=Functional+characterization+of+murine+interferon+regulatory+factor+5+(IRF-5)+and+its+role+in+the+innate+antiviral+response.">Functional characterization of murine interferon regulatory factor 5 (IRF-5) and its role in the innate antiviral response.</a> Journal of Biological Chemistry. 283 (21), 14295-14308 (2008).</li><li>Yasuda, K., 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=Murine+dendritic+cell+type+I+IFN+production+induced+by+human+IgG-RNA+immune+complexes+is+IFN+regulatory+factor+(IRF)5+and+IRF7+dependent+and+is+required+for+IL-6+production.">Murine dendritic cell type I IFN production induced by human IgG-RNA immune complexes is IFN regulatory factor (IRF)5 and IRF7 dependent and is required for IL-6 production.</a> The Journal of Immunology. 178 (11), 6876-6885 (2007).</li><li>Steinhagen, F., 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=IRF-5+and+NF-kappaB+p50+co-regulate+IFN-beta+and+IL-6+expression+in+TLR9-stimulated+human+plasmacytoid+dendritic+cells.">IRF-5 and NF-kappaB p50 co-regulate IFN-beta and IL-6 expression in TLR9-stimulated human plasmacytoid dendritic cells.</a> European Journal of Immunology. 43 (7), 1896-1906 (2013).</li><li>Gratz, 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=Type+I+interferon+production+induced+by+Streptococcus+pyogenes-derived+nucleic+acids+is+required+for+host+protection.">Type I interferon production induced by Streptococcus pyogenes-derived nucleic acids is required for host protection.</a> PLoS Pathogens. 7 (5), 1001345 (2011).</li></ol>

The authors have nothing to disclose.

The protocol described here is a modified native PAGE that distinguishes both monomeric and dimeric forms of endogenous IRF5. There have been few studies reporting the detection of endogenous IRF5 activation using the specialized imaging flow cytometry technique23,27,28,30. This protocol uses a common technique and commonplace reagents and tools to assess the endogenous IRF5 activation state du...

Interferon regulatory factor 5 (IRF5) is an important transcription regulator that plays a prominent role in regulating the immune response, particularly in the release of pro-inflammatory cytokines and type I interferons (IFNs)1,2,3. Misregulation of IRF5 is a contributing factor in numerous autoimmune diseases, as evident by various polymorphisms in the IRF5 locus that are associated with systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, etc.4,5,6,7,8,9,10. Therefore, a robust detection assay for endogenous IRF5 activation state is crucial for understanding the regulatory pathways and downstream effects of IRF5 in a physiologically relevant cellular context.
IRF5 is constitutively expressed in monocytes, dendritic cells (DCs), B cells, and macrophages1,11. As with other IRF family transcription factors, IRF5 resides in the cytoplasm in its latent state. Upon activation, IRF5 is phosphorylated and forms homodimers, which then translocate into the nucleus and bind to specific regulatory elements of genes encoding type I IFNs and pro-inflammatory cytokines, eventually inducing the expression of these genes1,2,11,12,13. IRF5 regulates the innate immune responses downstream of various Toll-like receptors (TLRs), such as TLR7, TLR 8, and TLR 9, which are localized in endosomes and use MyD88 for signaling1,11,14. These TLRs primarily recognize foreign nucleic acid species such as single-stranded RNA (ssRNA) and unmethylated CpG DNA that are symptomatic of an infection15,16,17,18. IRF5 has been shown to regulate immune responses against bacterial, viral, and fungal infections19,20,21. Considering IRF5&rsquo;s influential and diverse role in the immune system, enhancing or dampening IRF5 activity could serve as a novel avenue for the development of therapeutic agents22. Hence, it is critical to develop a protocol to monitor the activation status of endogenous IRF5 to allow thorough investigation of the pathways and mechanisms regulating IRF5 activity in different cell types.
To the best of our knowledge, no biochemical or gel electrophoretic assay for endogenous IRF5 activation has been published prior to the development of this protocol. Phosphorylation has been shown to be an important first step of IRF5 activation, and a phosphospecific IRF5 antibody was developed that led to the discovery and confirmation of a serine residue important for IRF5 activity13. However, while the antibody clearly detects phosphorylated IRF5 when immunoprecipitated or overexpressed23, it fails to detect IRF5 phosphorylation in a whole cell lysate in our hands (data not shown). Dimerization is the next step of IRF5 activation, and many important studies to date investigating this step relied on overexpression of epitope-tagged IRF5, often in irrelevant cell types that do not normally express IRF511,12,24,25. Previous studies have shown that dimerized IRF5 may not always translocate into the nucleus and hence is not necessarily fully activated25,26. An assay for endogenous IRF5 nuclear localization was developed to assess IRF5 activation by imaging flow cytometry27. This assay has been applied in studies that were crucial to understanding IRF5 activity, especially in primary or rare cell types28,29 and greatly advanced the knowledge in the field. However, this assay relies on a specialized instrument that is not widely available to researchers. Further, it is often necessary to investigate the initial steps of activation while dissecting IRF5 regulatory pathways and identifying upstream regulators and pathway components. This study provides a robust and reliable biochemical assay for the early activation events of IRF5 that can be performed in labs equipped with molecular biology tools. The protocol described here will be very useful in investigating the pathways and mechanisms of IRF5 actions, especially when combined with orthogonal assays such as the imaging flow cytometric analysis of IRF5 nuclear localization23,27,28,30.
Native polyacrylamide gel electrophoresis (native PAGE) is a widely used method to analyze protein complexes31,32. Unlike sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE separates proteins on the basis of their shape, size, and charge. It also retains native protein structure without denaturation31,33,34,35. The protocol presented takes advantage of these features of native PAGE and detects both monomeric and dimeric forms of IRF5. This method is particularly important for detecting early activation events because there is no suitable commercially available antibody that can detect endogenous phosphorylated IRF5. Previously, several published studies used native PAGE to assess IRF5 dimerization. However, the majority of these studies depended on the overexpression of exogenous epitope-tagged IRF5 to analyze activation status2,13,24,36,37. This work presents a step-by-step protocol for analyzing endogenous IRF5 dimerization via a modified native PAGE technique in a human plasmacytoid dendritic cell (pDC) line, where IRF5 activity has been shown to be crucial for its function1,38,39,40. This same technique has been applied to other cell lines23.

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Life Technologies, HK</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>300 W/250 V power supply 230 V AC</td>
			<td>Life Technologies, HK</td>
			<td>PS0301</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-IRF5 antibody</td>
			<td>Bethyl Laboratories, USA</td>
			<td>A303-385</td>
			<td></td>
		</tr>
		<tr>
			<td>BIOSAN Rocker Shaker (cold room safe)</td>
			<td>EcoLife, HK</td>
			<td>MR-12</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA Buffer, pH 8, 0.5 M 4 X 100 mL</td>
			<td>Life Technologies</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol 500 mL</td>
			<td>Life Technologies</td>
			<td>15514011</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Life Technologies, HK</td>
			<td>15527013</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG DyLight 800 Conjugated Antibody</td>
			<td>LAB-A-PORTER/Rockland, HK</td>
			<td>610-145-002-0.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG DyLight 800 Conjugated Antibody</td>
			<td>LAB-A-PORTER/Rockland, HK</td>
			<td>611-145-002-0.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt protease inhibitor cocktail (100x)</td>
			<td>Thermo Fisher Scientific, HK</td>
			<td>78430</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Life Technologies, HK</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>LI-COR Odyssey Blocking Buffer (TBS)</td>
			<td>Gene Company, HK</td>
			<td>927-50000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Tank blot module combo; Transfer module, accessories</td>
			<td>Life Technologies, HK</td>
			<td>NW2000</td>
			<td></td>
		</tr>
		<tr>
			<td>NativePAGE 3-12% gels, 10 well kit</td>
			<td>Life Technologies, HK</td>
			<td>BN1001BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>NativePAGE Running Buffer 20x</td>
			<td>Life Technologies, HK</td>
			<td>BN2001</td>
			<td></td>
		</tr>
		<tr>
			<td>NativePAGE Sample Buffer 4x</td>
			<td>Life Technologies, HK</td>
			<td>BN2003</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40 Alternative, Nonylphenyl Polyethylene Glycol</td>
			<td>Tin Hang/Calbiochem, HK</td>
			<td>#492016-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 7.4</td>
			<td>Life Technologies, HK</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylidene difluoride (PVDF) membrane</td>
			<td>Bio-gene/Merck Millipore, HK</td>
			<td>IPFL00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein assay kit II (BSA)</td>
			<td>Bio-Rad, HK</td>
			<td>5000002</td>
			<td></td>
		</tr>
		<tr>
			<td>R848</td>
			<td>Invivogen, HK</td>
			<td>tlrl-r848</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life Technologies, HK</td>
			<td>61870127</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>ThermoFisher</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate &#8805;97% (titration)</td>
			<td>Tin Hang/Sigma, HK</td>
			<td>D6750-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Life Technologies, HK</td>
			<td>15504020</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Tin Hang/Sigma, HK</td>
			<td>#P9416-100ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

native polyacrylamide gel electrophoresis immunoblot analysis of endogenous irf5 dimerization

Interferon regulatory factor 5 (IRF5) is a key transcription factor for regulating the immune response. It is activated downstream of the Toll-like receptor myeloid differentiation primary response gene 88 (TLR-MyD88) signaling pathway. IRF5 activation involves phosphorylation, dimerization, and subsequent translocation from the cytoplasm into the nucleus, which in turn induces the gene expression of various pro-inflammatory cytokines. A detection assay for IRF5 activation is essential to studying IRF5 functions and its relevant pathways. This article describes a robust assay to detect endogenous IRF5 activation in the CAL-1 human plasmacytoid dendritic cell (pDC) line. The protocol consists of a modified nondenaturing electrophoresis assay that can distinguish IRF5 in its monomer and dimer forms, thus providing an affordable and sensitive approach to analyze IRF5 activation.

The immunoblot (IB) with an anti-IRF5 antibody was performed on CAL-1 cells unstimulated or stimulated with 1 &#181;g/mL R848 for 2 h (Figure 1). Cell lysates were prepared, and the native PAGE was performed. In unstimulated CAL-1 cells, IRF5 was detected as a single band on the native PAGE, corresponding to its monomeric form. Upon treatment of CAL-1 cells with R848 for 2 h, the level of IRF5 monomer decreased with a concurrent increase in the accumulation of a slowly migrating band that co...

Watch this Scientific Journal Video about Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization at JoVE.com

Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization

A native Western blot method for analyzing endogenous interferon regulatory factor 5 dimerization in the CAL-1 plasmacytoid dendritic cell line is described. This protocol can be applied to other cell lines as well.

Interferon regulatory factor 5 (IRF5) is a key transcription factor for regulating the immune response. It is activated downstream of the Toll-like ...

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9.8K Views.  City University of Hong Kong. A native Western blot method for analyzing endogenous interferon regulatory factor 5 dimerization in the CAL-1 plasmacytoid dendritic cell line is described. This protocol can be applied to other cell lines as well.

Video: Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...