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>

<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., 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 activat...

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 border="0" cellpadding="0" cellspacing="0">
	<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&#8217;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>Six-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&#8211;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 &#956;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 &#956;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>20g and 23g 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>Six-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&#8211;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 &#956;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 &#956;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>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 macr...

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

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JoVE Journal

Immunology and Infection

35.9K 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.