This work was supported by a grant from the&#160;Natural Sciences and Engineering Research Council of Canada (NSERC, grant no 298515-2011).&#160;AL is the recipient of a Ph.D. scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC),&#160;and MS was supported from a grant from the Canadian Institutes of Health Research (CIHR, grant no. MOP123246).

All procedures were performed in accordance with the Canadian Council on Animal Care guidelines after approval by the institutional Animal Care Committee of the Centre de recherche du Centre Hospitalier de l&#8217;Universit&#233; de Montr&#233;al (CRCHUM).
1. Isolation, Identification, and Culture of Murine Peritoneal Macrophages
<ol>
	<li>Prepare 3.8% brewer&#8217;s thioglycollate medium. To do so, suspend 38 g of thioglycollate medium in 1,000 ml of distilled water. Bring solution to boil to dissolve the medium completely. Sterilize by autoclaving at 121 &#176;C for 15 min. Store up to 3 months in the dark, at RT18. 
	&#8203;NOTE: Discard if turbidity develops which indicates a bacterial contamination. The solution can be kept for up to one year in the dark if kept sterile.</li>
	<li>Using 1 ml syringes attached to 23 G needles, inject 1 ml of 3.8% Brewer thioglycollate medium into the peritoneal cavity of each mouse and wait for 3 days. Use new syringe and needle for each mouse.</li>
	<li>Anesthetize mice by intraperitoneal injection of sodium pentobarbital (100 mg/kg) and euthanize mice by cervical dislocation. Confirm proper anesthetization by checking the respiratory rate. Usually rapid respirations indicate that the mouse is not deeply anaesthetized.</li>
	<li>Wash the abdomen of each mouse with 70% ethanol. Using a scissor, perform a lateral incision along the bottom midline of the peritoneum.</li>
	<li>Using a forceps, pull back the abdominal skin to expose the transparent peritoneal skin. Using 5 ml syringes attached to 20 G needles, inject 5 ml of cold DPBS into the peritoneal cavity of each mouse.</li>
	<li>Perform a gentle massage on the peritoneal cavity and then aspirate the fluid carefully without puncturing any organ. Remove the needle and dispense the peritoneal fluid into 50 ml conical centrifuge tubes.</li>
	<li>Centrifuge for 10 min at 400 x g in a refrigerated centrifuge. Cells must stay cold during the whole procedure. Discard supernatant and resuspend cell pellet in RPMI medium 1640.</li>
	<li>Using a hemocytometer, count cells&#160;and adjust to cell density to 1 x 106&#160;cells/ml.&#160;</li>
	<li>Characterize the phenotype of isolated cells by flow cytometry using 1 x 106 cells per mouse and antibodie against F4/80 (a surface antigen&#160;expressed on macrophages).</li>
</ol>
2. Cell Treatments
<ol>
	<li>Directly, after isolation, add 1 x 106&#160;cells into each well. Leave the murine peritoneal macrophages to adhere into the 6-well plates by culturing them for 1 to 2 hr at 37 &#176;C. Remove non-adherent cells by gently washing 3 times with warm PBS.</li>
	<li>Subsequently, culture cells in 900 &#181;l serum-free DMEM for 24 hr in the presence of the following TLR ligands: (Pam3CSK4-TLR1/2 (0.5 mg/ml); Poly(I:C)-TLR3 (10 mg/ml); LPS-TLR4 (100 ng/ml); Flagellin-TLR5 (100 ng/ml); FSL1-TLR6/2 (100 ng/ml); ssRNA40-TLR7 (1 &#181;g/ml); ODN1826-TLR9 (1 &#181;M). 
	NOTE: Prepare for each ligand a 10x stock solution. Add 100 &#181;l of the latter to each well, thus performing a 10-fold dilution.</li>
</ol>
3. RNA Isolation
<ol>
	<li>Remove all medium and lyse cells directly in the six-well plates by adding 1 ml TRIzol to each well and passing the cell lysate several times through a pipette. Incubate the homogenised samples for 5 to 10 min at RT, to allow the complete dissociation of nucleoprotein complexes.</li>
	<li>Transfer lysate into 1.5 ml RNase- and DNase-free microcentrifuge tubes. Add 0.2 ml chloroform per 1ml TRIzol. Shake tubes vigorously by hand for 15 sec and incubate them at RT for 5 to 10 min. Centrifuge at 12,000 x g for 15 min at 4 &#176;C. Observe the mixture separating into a lower red phase (phenol-chloroform), an interphase and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase.</li>
	<li>Transfer carefully the upper aqueous phase to a fresh tube without disturbing the interphase. Precipitate the RNA from it by mixing with 0.5 ml of isopropyl alcohol. Incubate samples at RT for 10 min and centrifuge at 12,000 x g for 10 min at 4 &#176;C. The RNA precipitates forming a white pellet on the side and bottom of the tube.</li>
	<li>Remove supernatant. Wash the RNA pellet once with 1 ml of 75% ethanol. Mix the samples by vortexing and centrifuge at 7,500 x g for 5 min at 4 &#176;C. Briefly dry the RNA (air dry for 10 min). Dissolve RNA in 0.1 ml DEPC (RNAse free water) and incubate for 10 min at 55 &#176;C.</li>
	<li>Quantify RNA using a spectrophotometer. To do so, dilute samples 100 times in DEPC water and read at wavelengths of 260 nm. The RNA concentration will then be: OD260 x 40 ng/ul x dilution factor.</li>
	<li>Equalize RNA concentrations and synthesize&#160;cDNA as per manufacturer&#8217;s instructions using RT-PCR System for First-Strand cDNA Synthesis Kit. As per manufacturer&#8217;s instructions, measure gene mRNA levels by real-time PCR (45 cycles) in a Real-Time DNA detection system. Normalize gene expression levels with two housekeeping gene for example &#946;-actin and GAPDH. 
	NOTE: Normalize RNA concentration to 500 &#181;g/ml and use 0.5 &#181;g for cDNA synthesis.</li>
</ol>

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F., Ponka, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-heme+induction+of+heme+oxygenase-1+does+not+alter+cellular+iron+metabolism.">Non-heme induction of heme oxygenase-1 does not alter cellular iron metabolism.</a> J Biol Chem. 282 (14), 10480-10486 (2007).</li><li>Hershko, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Storage+iron+regulation.">Storage iron regulation.</a> Prog Hematol. 10, 105-148 (1977).</li><li>Collins, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Withholding+iron+as+a+cellular+defence+mechanism--friend+or+foe.">Withholding iron as a cellular defence mechanism--friend or foe.</a> Eur J Immunol. 38 (7), 1803-1806 (2008).</li><li>Noyes, W. D., Bothwell, T. H., Finch, C. 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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=Toll-like+receptor+signal+adaptor+protein+MyD88+is+required+for+sustained+endotoxin-induced+acute+hypoferremic+response+in+mice.">Toll-like receptor signal adaptor protein MyD88 is required for sustained endotoxin-induced acute hypoferremic response in mice.</a> Am J Pathol. 180 (6), 2340-2350 (2012).</li><li>Zumerle, S., 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=Targeted+disruption+of+hepcidin+in+the+liver+recapitulates+the+hemochromatotic+phenotype.">Targeted disruption of hepcidin in the liver recapitulates the hemochromatotic phenotype.</a> Blood. 123 (23), 3646-3650 (2014).</li><li>Nguyen, N. B., Callaghan, K. D., Ghio, A. J., Haile, D. J., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepcidin+expression+and+iron+transport+in+alveolar+macrophages.">Hepcidin expression and iron transport in alveolar macrophages.</a> Am J Physiol Lung Cell Mol Physiol. 291 (3), L417-L425 (2006).</li><li>Nemeth, E., 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=Hepcidin,+a+putative+mediator+of+anemia+of+inflammation,+is+a+type+II+acute-phase+protein.">Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein.</a> Blood. 101 (7), 2461-2463 (2003).</li><li>Nemeth, E., 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=Hepcidin+regulates+cellular+iron+efflux+by+binding+to+ferroportin+and+inducing+its+internalization.">Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.</a> Science. 306 (5704), 2090-2093 (2004).</li><li>Constante, 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=Distinct+requirements+for+Hfe+in+basal+and+induced+hepcidin+levels+in+iron+overload+and+inflammation.">Distinct requirements for Hfe in basal and induced hepcidin levels in iron overload and inflammation.</a> Am J Physiol Gastrointest Liver Physiol. 291 (2), G229-G237 (2006).</li><li>Zhang, X., Goncalves, R., Mosser, D. 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N., van Furth, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+thioglycolate+on+phagocytic+and+microbicidal+activities+of+peritoneal+macrophages.">Effect of thioglycolate on phagocytic and microbicidal activities of peritoneal macrophages.</a> Infect Immun. 46 (2), 448-452 (1984).</li><li>Dy, M., Debray-Sachs, M., Kamoun, P., Hamburger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+thioglycollate+on+macrophage+lysosomal+enzymes.">Effect of thioglycollate on macrophage lysosomal enzymes.</a> Biomedicine. 29 (5), 167-170 (1978).</li><li>Li, Y. M., Baviello, G., Vlassara, H., Mitsuhashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycation+products+in+aged+thioglycollate+medium+enhance+the+elicitation+of+peritoneal+macrophages.">Glycation products in aged thioglycollate medium enhance the elicitation of peritoneal macrophages.</a> J Immunol Methods. 201 (2), 183-188 (1997).</li><li>Layoun, A., Santos, 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=Bacterial+cell+wall+constituents+induce+hepcidin+expression+in+macrophages+through+MyD88+signaling.">Bacterial cell wall constituents induce hepcidin expression in macrophages through MyD88 signaling.</a> Inflammation. 35 (4), 1500-1506 (2012).</li><li>Oppenheim, J. J., Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alarmins:+chemotactic+activators+of+immune+responses.">Alarmins: chemotactic activators of immune responses.</a> Curr Opin Immunol. 17 (4), 359-365 (2005).</li><li>Akira, S., Uematsu, S., Takeuchi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+recognition+and+innate+immunity.">Pathogen recognition and innate immunity.</a> Cell. 124 (4), 783-801 (2006).</li><li>Lang, R., Patel, D., Morris, J. J., Rutschman, R. L., Murray, P. 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The authors have no competing financial interests.

Macrophages are crucial for survival and provide a tempting target to manipulate the host for immunological objectives. The discovery of TLRs and other recognition molecules have conducted the macrophages to the centre of immunological debate. Macrophages respond to a variety of stimuli, including cytokines, damage-associated molecular pattern molecules (DAMPs)20 and molecules associated with groups of pathogens (PAMPs)21. These different stimuli responses represent the course of macrophages activation, and are usually associated with sudden alterations in gene expression22.
In non-inflammatory conditions, the majority of macrophages reside in strategic locations in the body. They can be found in all tissues and as circulating monocytes in the blood. Therefore, macrophages are present in the most susceptible sites for microbial invasion.
The host defense is described as an inflammatory response to the elimination of intracellular pathogens associated with classical macrophage activation. The classical activation of macrophages is induced by microbial products such as lipopolysaccharides in a Th1 cytokine environment which will lead to pro-inflammatory M1-macrophages polarization. The persistence of inflammation results in tissue damage and the development of anti-inflammatory mechanisms necessary to the survival of the host. Therefore, Th2 cytokines allow then the introduction of anti-inflammatory M2-macrophages polarization which inhibits and regulates the M1 response, and also promotes tissue repair. In the protocol described in this paper, thioglycollate injection in the peritoneal cavity stimulates the classical inflammatory cascade and leads to the recruitment of M1 macrophages.
To date, several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. These macrophages represent heterogeneous populations with different activities. Based on their morphology and surface molecular characteristics, studies have established that peritoneal macrophages are more mature than bone marrow and spleen derived macrophages23. Unlike spleen and peritoneum derived macrophages, bone marrow derived macrophages present a remarkable ability in pahgocytosis and proliferation and can be completely differentiated from macrophage progenitor cells20,24,25. In addition, the isolation of bone marrow macrophages presents a homogenous yield with long lifespan. On the other hand, these macrophages are not fully characterized and their use in experimental studies presents complications due to the inconstancy of their phenotype and functions26 . Unlike bone marrow derived macrophages, spleen and peritoneum macrophages seem to be more functionally and phenotypically stable23 .
Hence, the isolation of murine peritoneal macrophages can serve different immunological studies and gene expression analysis. In addition, the mouse peritoneal cavity affords an ideal site for harvesting resident macrophages27. However, the elicited number is moderate and estimated around 1 x 106&#160;macrophages per mouse. Thus, to increase the harvest of macrophages, eliciting agents such as thioglycollate was injected in the peritoneal cavity 3 days before cell isolation28. This agent will induce an inflammatory response and accordingly increase macrophage crop.
It&#8217;s imperative to perform a gentle massage on the peritoneal cavity before withdrawing slowly the peritoneal fluid without puncturing any organ. Pulling out the maximal possible volume is required to collect the largest cell number. In case of blood contamination, a lysis buffer can be used at the end of the procedure to discard red blood cells. The elicited macrophages can then be characterized by flow cytometry using antibodies against antigens that are unique to&#160;macrophages as F4/80.
Throughout the procedure, make sure that all reagents are endotoxin-free and all animals were permanently housed under specific pathogen-free conditions. Performing this procedure under pathogen-free conditions is critical because macrophage stimulation prior to impending experiments will alter significantly the results analysis.
Once isolated, the peritoneal macrophages can be used in several studies, including production of inflammatory cytokines, phagocytosis, cell signaling, gene expression, chemotaxis and toxicology29. For example, after stimulation with different TLR ligands, we investigated in the elicited macrophages the molecular regulation of a key regulator of iron metabolism named hepcidin. 24 hr after cell treatments, total RNA was isolated with TRIzol reagent, and gene expression was analysed using qRT-PCR.
As we are studying TLRs activation and gene expression, the use of serum free DMEM is recommended. Serum free medium reduces the degree of contaminants and eliminate any potential source of infectious agents.
Albeit RNA isolation seems a simple process, RNase and DNA contamination must be avoided to prevent RNA degradation and achieve accurate qRT-PCR results. The best way to detect DNA contamination is to include a &#39;minus-RT&#39; control for each RNA sample in an RT-PCR experiment. If a PCR product is generated from an RNA sample that was not reverse transcribed then the product was amplified from contaminating DNA.&#160;In case of DNA contamination, the use of DNase treatment is possible. However, DNase must be completely inactivated prior to RT-PCR so that it doesn&#39;t degrade newly synthesized DNA.

The reticuloendothelial phagocytic system is composed of cells in various tissues and organs such as bone marrow, blood, liver and spleen. Macrophages are extensively distributed around the body, where they notably participate in innate and adaptive immune responses to control and clear infections. In addition to their role in host defense, macrophages also play an important role in wound healing and in maintaining tissue homeostasis1,2. Furthermore, macrophages are not only important to immune function but also actively participate in iron homeostasis3. In the body, approximately 80% of iron is present in hemoglobin within erythrocytes, which when senescent are phagocytosed by macrophages4. Daily, these macrophages recycle 25 mg of erythrocyte-derived iron and provide its transport into the plasma5. Moreover, during infection and inflammation, pro-inflammatory macrophages sequester serum iron to reduce iron availability to pathogens, at both the systemic and local levels6-8. As well, studies have shown that macrophages and mainly hepatocytes produce an antimicrobial peptide named hepcidin that is considered the master&#160;regulator of&#160;iron&#160;metabolism9,10. Hepcidin is mainly increased by inflammatory stimuli and is partially responsible for iron sequestration in macrophage upon chronic inflammation11-13.&#160;As hepcidin expression in macrophages is not very well understood, we studied the possible role of Toll-like receptors (TLRs) in this regulation. The TLRs&#160;are primarily found on&#160;macrophages and play a central role in&#160;their activation. In addition, LPS induced hepcidin expression in the liver is dependent on TLR413. Therefore, to execute our study, we used a method based on the isolation of murine peritoneal macrophages.
Macrophage cell lines are broadly used in macrophage studies; nonetheless extended culture can provoke gene loss and decreased immune functions in these cell lines. Thus, isolation of macrophages from peritoneal cavity is crucial.
The mouse peritoneal cavity presents an ideal site to harvest macrophages13-15. Isolated murine peritoneal macrophages are convenient for several studies regarding their immunological function. However, the number of macrophages in the peritoneum is insufficient for extensive studies and is estimated around 1 x 106 macrophages per mouse. Thus, to raise macrophage output, a sterile eliciting agent such as thioglycollate was injected into the peritoneal cavity preceding the cell harvest. After thioglycollate injection, the yield of macrophages per mouse was increased by 10-fold. Despite the increase in macrophages yield, Brewer&#8217;s thioglycollate medium acts as an irritant that induces an inflammatory response, resulting in the recruitment of macrophages, which may but not necessary affect gene expression. Hence, a control group consisting of non-treated macrophages must be included in each experiment. In our hands, hepcidin expression which is highly stimulated by inflammation was not detected in non-treated thioglycollate elicited peritoneal macrophages. Moreover, studies have shown that Brewer&#8217;s thioglycollate recruits numerous macrophages, but does not activate them16. On the other hand, Brewer&#8217;s thioglycollate elicited macrophages showed an increase in lysosomal enzyme but a decrease in&#160;killing ingested microorganisms17. However, the phagocytic capacity was not affected when compared with non-elicited macrophages16.
Once cultured in dishes, the peritoneal macrophages become adherent, therefore allowing their separation from other type of cells isolated from the peritoneal cavity. Subsequently, the isolated macrophages were challenged with different TLRs agonists. Finally, mRNA was extracted&#160;from the cultured&#160;cells and gene expression was analysed using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).

<table><tbody><tr><td>C57BL/6 mice</td><td>Charles River Laboratories, Inc. (Wilmington, MA, USA)</td><td>475</td><td></td></tr><tr><td>Thioglycollate</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>19032-500G</td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>10% sodium pentobarbital (Used for mice anesthesia (80 mg/kg, i.p.))</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate-buffered saline (DPBS), placed on ice and will serve to harvest macrophages</td><td>WISENT INC Canada (QC)</td><td>311-425-CL</td><td></td></tr><tr><td>RPMI medium 1640 (Supplement with penicillin, streptomycin, L-glutamine, and 10 % fetal calf serum).</td><td>WISENT INC Canada (QC)</td><td>350-000-CL</td><td></td></tr><tr><td>1 and 5 ml syringes</td><td>BD USA (NJ)</td><td>309659</td><td></td></tr><tr><td>6-well plates</td><td>Corning Incorporated (NY, USA)</td><td>MCT-150-C</td><td></td></tr><tr><td>Bacterial lipoprotein Pam3CSK4 (0.5 mg/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLRL-pm25</td><td></td></tr><tr><td>Polyionosine&amp;ndash;polycytidylic acid (Poly(I:C)) (10 mg/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLRL-PIC</td><td></td></tr><tr><td>LPS from Escherichia coli 055:B5 (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>L2880</td><td></td></tr><tr><td>Purified flagellin from Salmonella typhimurium (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-FLIC-10</td><td></td></tr><tr><td>Lipoprotein synthetic FSL1 (100 ng/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-FSL</td><td></td></tr><tr><td>ssRNA derived from the HIV-1 long terminal repeat ssRNA40 (1 &amp;mu;g/ml)</td><td>InvivoGen (San Diego, USA)</td><td>TLR-LRNA-40</td><td></td></tr><tr><td>Type B CpG oligonucleotide ODN1826 (1 &amp;mu;M)</td><td>InvivoGen (San Diego, USA)</td><td>11B16-MM</td><td></td></tr><tr><td>TRIzol</td><td>Invitrogen, (Burlington, ON, Canada)</td><td>15596-026</td><td></td></tr><tr><td>20 G and 23 G needles</td><td>BD USA (NJ)</td><td>305175</td><td></td></tr><tr><td>Scissor</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>50 ml conical tubes placed on ice</td><td>Sarstedt (Newton, MA, USA)</td><td>62.547.205</td><td></td></tr><tr><td>Red Blood Cells Lysis Buffer</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>R7757-100ML</td><td></td></tr><tr><td>Refrigerated centrifuge</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>F4/80 antibody</td><td>BIO-RAD ( CA, USA)</td><td>MCA497APC</td><td></td></tr><tr><td>CD16/CD32 antibodies</td><td>Pharmingen, {Mississauga, ON, CA)</td><td>553141</td><td></td></tr><tr><td>Flow cytometer</td><td>Coulter Epics Elite counter, Coulter, (Hialeah, FL,USA)</td><td></td><td></td></tr><tr><td>1.5 ml Eppendorf tubes</td><td>Axygen Scietific (CA,USA)</td><td>3516</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific (ON, Canada)</td><td>UN1888</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>JT Baker (PA, USA)</td><td>70566</td><td></td></tr><tr><td>75% ethanol (in DEPC treated water)</td><td>Commercial Alchohols (QC, Canada)</td><td>17394</td><td></td></tr><tr><td>0.01% diethyl pyrocarbonate (DEPC) treated water (let stand overnight and autoclave)</td><td>Sigma-Aldrich, (St. Louis, MO)</td><td>216.542.8</td><td></td></tr><tr><td>Omniscript RT-PCR system</td><td>Qiagen, (Mississauga, ON, Canada)</td><td>205113</td><td></td></tr><tr><td>Rotor Gene 3000</td><td>Montreal Biotech, (Kirkland, QC, Canada)</td><td></td><td></td></tr><tr><td>QuantiTect SYBR Green I PCR kits</td><td>Qiagen, (Mississauga, ON, Canada)</td><td>204141</td><td></td></tr></tbody></table>

isolation of murine peritoneal macrophages to carry out gene expression analysis upon toll-like receptors stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We first characterized the isolated murine peritoneal macrophages by flow cytometry. To do so, we used (F4/80) antibodies that specifically recognize markers only expressed by macrophages. This characterization is required to determine the percentage of isolated macrophage and to distinguish them among cells obtained during the isolation process. As shown in (Figure 1), the percentage of cells expressing the antigen F4/80 was consistently found to be above 95%. Next, to study&#160;gene expression in macrophages, the isolated cells were treated with several TLR ligands: Pam3CSK4 (TLR1/2), LPS (TLR4) and FSL1 (TLR6/2). Subsequently, mRNA levels of Hepcidin (Hamp), our gene of interest, were measured by RT-PCR. As shown in (Figure 2), TLR1/2, TLR4 and TLR6/2 ligands were capable of stimulating hepcidin mRNA in murine macrophages19. Together, these results demonstrate the usefulness of this protocol to successfully isolate murine peritoneal macrophages and to investigate precisely the molecular regulation of gene expression.
<img alt="Figure 1" src="/files/ftp_upload/52749/52749fig1.jpg" /> 
Figure 1. Characterisation of cells isolated from the peritoneal cavity. Enrichment of the recovered macrophages was confirmed by flow cytometric analysis using the F4/80 antibody after blocking nonspecific staining with CD16/CD32 antibodies and was consistently found to be above 95%.
<img alt="Figure 2" src="/files/ftp_upload/52749/52749fig2.jpg" /> 
Figure 2. TLR ligands induce hepcidin expression in murine peritoneal macrophages.&#160;After murine peritoneal macrophages isolation and stimulation with TLR1/2, TLR4 and TLR6/2 ligands, hepcidin mRNA levels were studied by quantitative reverse transcriptase-polymerase chain reaction. Data are presented as mean &#177; SEM; n.d. (not detectable); *P &#60; 0.05 versus control (Ctrl). Results are representative of 3 similar experiments performed independently.

Watch this Scientific Journal Video about Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation at JoVE.com

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

isolation-murine-peritoneal-macrophages-to-carry-out-gene-expression

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

36.2K Views.  Institut du cancer de Montréal. We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Video: Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Cancer Research

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Medicine

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 ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

Analysis of Efferocytosis of Apoptotic Thymocytes by Peritoneal Macrophages

Source: Zhen, Y. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59731">Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages.</a> J. Vis. Exp. (2019)
This video demonstrates a procedure for analyzing peritoneal macrophage-mediated efferocytosis of apoptotic mouse thymocytes by fluorescence microscopy.

Source: Zhen, Y. et al., Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages. J. Vis. Exp. (2019)
This video demonstrates a procedure ...