Questo lavoro è stato sostenuto da una sovvenzione da parte scienze naturali e ingegneria Research Council del Canada (NSERC, concedere senza 298515-2011). AL è il destinatario di un dottorato di ricerca borsa di studio le scienze naturali e ingegneria Research Council del Canada (NSERC), e MS è stato sostenuto da una sovvenzione da parte del Canadian Institutes of Health Research (CIHR, concessione n. MOP123246).

Tutte le procedure sono state eseguite in accordo con il Consiglio canadese sugli orientamenti per la cura degli animali, dopo l&#39;approvazione da parte del Comitato istituzionale cura degli animali del Centro de recherche du Centre Hospitalier de l&#39;Université de Montréal (CRCHUM). 1. Isolamento, identificazione, e Cultura Murine Peritoneale macrofagi <ol><li> Preparare medio tioglicollato 3,8% di birra. Per fare ciò, sospendere 38 g di tioglicollato medie 1.000 ml di acqua distillata. Portare la soluzione ad ebollizione per sciogliere completamente il mezzo. Sterilizzare in autoclave a 121 ° C per 15 min. Memorizza fino a 3 mesi al buio, a temperatura ambiente 18. NOTA: Eliminare se si sviluppa torbidità che indica una contaminazione batterica. La soluzione può essere conservato fino ad un anno al buio se mantenuto sterile. </li><li> Uso 1 ml siringhe allegate a 23 aghi G, iniettare 1 ml di 3,8% Brewer tioglicolato medio nella cavità peritoneale di ogni mouse e attendere per 3 giorni. Utilizzare nuova siringa e l&#39;ago per ogni mouse. </li><li> Anestetizzare topi mediante iniezione intraperitoneale di sodio pentobarbital (100 mg / kg) e eutanasia topi mediante dislocazione cervicale. Verificare il corretto anestesia controllando la frequenza respiratoria. Di solito respirazione rapida indicano che il mouse non è profondamente anestetizzato. </li><li> Lavare l&#39;addome di ciascun topo con il 70% di etanolo. Usando una forbice, effettuare un&#39;incisione laterale lungo la linea mediana inferiore del peritoneo. </li><li> Usando una pinza, tirare indietro la pelle addominale per esporre la pelle peritoneale trasparente. Utilizzando 5 ml siringhe attaccate a 20 aghi G, iniettare 5 ml di DPBS freddi nella cavità peritoneale di ciascun topo. </li><li> Eseguire un leggero massaggio sulla cavità peritoneale e quindi aspirare accuratamente il liquido, senza pungere qualsiasi organo. Rimuovere l&#39;ago e dispensare il liquido peritoneale in 50 ml provette da centrifuga coniche. </li><li> Centrifugare per 10 min a 400 xg in una centrifuga refrigerata.Le cellule devono restare freddo durante l&#39;intera procedura. Gettare il surnatante e pellet cellulare risospendere in mezzo RPMI 1640. </li><li> Utilizzando un emocitometro, contare le cellule e regolare a densità cellulare di 1 x 10 6 cellule / ml. </li><li> Caratterizzare il fenotipo di cellule isolate mediante citometria di flusso con 1 x 10 6 cellule per mouse e antibodie contro F4 / 80 (un antigene di superficie espresso sui macrofagi). </li></ol> 2. trattamenti con le cellule <ol><li> Direttamente, dopo l&#39;isolamento, aggiungere 1 x 10 6 cellule in ciascun pozzetto. Lasciare i macrofagi peritoneali murini aderire nelle piastre da 6 pozzetti da loro coltura per 1 a 2 ore a 37 ° C. Rimuovere le cellule non aderenti lavando delicatamente per 3 volte con caldo PBS. </li><li> Successivamente, cellule di coltura in 900 microlitri DMEM privo di siero per 24 ore in presenza dei seguenti ligandi TLR: (Pam3CSK4-TLR1 / 2 (0,5 mg / ml); poli (I: C) -TLR3 (10 mg / ml) ; LPS-TLR4 (100 ng / ml), flagellina-TLR5 (100 ng / ml); FSL1-TLR6 / 2 (100 ng / ml); ssRNA40-TLR7 (1 mg / ml); ODN1826-TLR9 (1 micron). NOTA: Preparare per ogni legante una soluzione 10x. Aggiungere 100 ml di quest&#39;ultima in ogni pozzetto, eseguendo così una diluizione di 10 volte. </li></ol> 3. RNA Isolation <ol><li> Rimuovere tutte le cellule medie e Lisare direttamente nelle piastre a sei pozzetti aggiungendo 1 ml TRIzol a ciascun pozzetto e passando il lisato cellulare più volte attraverso una pipetta. Incubare i campioni omogeneizzati per 5 a 10 min a temperatura ambiente, per consentire la completa dissociazione dei complessi nucleoproteici. </li><li> Trasferire il lisato in 1,5 ml RNasi e microprovette senza DNasi. Aggiungere 0,2 ml di cloroformio per 1 ml TRIzol. Agitare tubi vigorosamente a mano per 15 secondi e incubare a temperatura ambiente per 5-10 minuti. Centrifugare a 12.000 xg per 15 min a 4 ° C. Osservare la miscela separa in una fase rossa (fenolo-cloroformio) inferiore, un interfase e una fase acquosa superiore incolore. RNA rimane esclusivamente nella fase acquosa. </li><li> TranSFER accuratamente la fase acquosa superiore in una nuova provetta senza disturbare l&#39;interfase. Precipitare il RNA da esso mescolando con 0,5 ml di alcool isopropilico. Incubare i campioni a temperatura ambiente per 10 min e centrifugare a 12.000 xg per 10 min a 4 ° C. L&#39;RNA precipitati formare una pallina bianca sul lato e fondo del tubo. </li><li> Rimuovere il surnatante. Lavare il RNA pellet una volta con 1 ml di etanolo al 75%. Miscelare i campioni di vortex e centrifugare a 7.500 xg per 5 minuti a 4 ° C. Brevemente asciugare l&#39;RNA (aria secca per 10 min). Sciogliere RNA in 0.1 ml di DEPC (RNAse acqua gratuita) e incubare per 10 minuti a 55 ° C. </li><li> Quantificare RNA utilizzando uno spettrofotometro. Per fare ciò, diluire i campioni di 100 volte in acqua DEPC e leggere a lunghezze d&#39;onda di 260 nm. La concentrazione di RNA sarà quindi: OD 260 x 40 ng / ul x fattore di diluizione. </li><li> Equalizza concentrazioni di RNA e sintetizzare cDNA secondo le istruzioni del fabbricante, con RT-PCR per First-Strand cDNA Synthesis Kit. Comele istruzioni del produttore, misurare i livelli di gene mRNA di real-time PCR (45 cicli) in un sistema di rilevazione del DNA in tempo reale. Normalizzare i livelli di espressione genica con due gene housekeeping ad esempio β-actina e GAPDH. NOTA: Normalizzare concentrazione di RNA a 500 mg / ml e 0,5 mg utilizzare per la sintesi del DNA. </li></ol>

<ol>
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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.

I macrofagi sono cruciali per la sopravvivenza e fornire un bersaglio allettante per manipolare l&#39;host per obiettivi immunologici. La scoperta di TLR e di altre molecole di riconoscimento hanno condotto i macrofagi al centro del dibattito immunologica. Macrofagi rispondono ad una varietà di stimoli, compresi citochine, molecole danni associati molecolari modello (smorza) 20 e molecole associate a gruppi di patogeni (PAMP) 21. Queste diverse risposte stimoli rappresentano corso dell&#39;attivaz...

Il sistema reticoloendoteliale fagocitica è composto di cellule in vari tessuti ed organi come il midollo osseo, sangue, fegato e milza. I macrofagi sono ampiamente distribuiti in tutto il corpo, dove in particolare partecipano innata e risposte immunitarie per il controllo e le infezioni chiare. Oltre al loro ruolo nella difesa dell&#39;ospite, macrofagi giocano un ruolo importante nella guarigione delle ferite e nel mantenimento dell&#39;omeostasi tissutale 1,2. Inoltre, i macrofagi non sono importanti solo per la funzione immunitaria, ma anche partecipare attivamente nell&#39;omeostasi del ferro 3. Nel corpo, circa il 80% di ferro è presente in emoglobina entro eritrociti, che quando sono senescente fagocitato dai macrofagi 4. Ogni giorno, i macrofagi riciclare 25 mg di ferro eritrociti di derivazione e di fornire il suo trasporto nel plasma 5. Inoltre, durante l&#39;infezione e l&#39;infiammazione, macrofagi pro-infiammatori sequestrano sideremia per ridurre DISPONIBIL ferroty agli agenti patogeni, sia a livello sistemico e locale 6-8. Inoltre, studi hanno dimostrato che i macrofagi e soprattutto epatociti producono un peptide antimicrobico denominato hepcidin che è considerato il principale regolatore del metabolismo del ferro 9, 10. Hepcidin è aumentato principalmente da stimoli infiammatori ed è parzialmente responsabile per il sequestro di ferro in macrofagi su infiammazione cronica 11-13. Come espressione di epcidina in macrofagi non è molto ben compreso, abbiamo studiato il possibile ruolo dei recettori Toll-like (TLR) nel presente regolamento. I TLR si trovano principalmente sui macrofagi e svolgono un ruolo centrale nella loro attivazione. Inoltre, LPS espressione hepcidin indotta nel fegato è dipendente TLR4 13. Pertanto, per eseguire il nostro studio, abbiamo usato un metodo basato sull&#39;isolamento dei macrofagi peritoneali murini. Linee di cellule macrofagi sono ampiamente utilizzati in stallone macrofagiies; tuttavia la cultura estesa può provocare la perdita di geni e diminuzione delle funzioni immunitarie in queste linee cellulari. Pertanto, l&#39;isolamento dei macrofagi dalla cavità peritoneale è cruciale. La cavità peritoneale del mouse presenta un luogo ideale per raccogliere i macrofagi 13-15. Isolati macrofagi peritoneali murini sono convenienti per diversi studi riguardanti la loro funzione immunologica. Tuttavia, il numero di macrofagi nel peritoneo è insufficiente per studi approfonditi ed è stimata intorno a 1 x 10 6 macrofagi per topo. Pertanto, per aumentare la produzione di macrofagi, un agente suscitando sterile come tioglicolato stato iniettato nella cavità peritoneale precedente il raccolto cella. Dopo l&#39;iniezione tioglicolato, la resa dei macrofagi per mouse è stato aumentato di 10 volte. Nonostante l&#39;aumento dei macrofagi rendimento, Thioglycollate atti medio di birra come irritante che induce una risposta infiammatoria, con conseguente reclutamento di macrofagi, che may ma non necessario influenzano l&#39;espressione genica. Quindi, un gruppo di controllo costituito da macrofagi non trattata deve essere incluso in ogni esperimento. Nelle nostre mani, espressione di epcidina che è altamente stimolata da infiammazione non è stato rilevato in Thioglycollate non trattato suscitato macrofagi peritoneali. Inoltre, gli studi hanno dimostrato che il tioglicollato di birra recluta numerosi macrofagi, ma non attiva le 16. D&#39;altra parte, tioglicolato di Brewer suscitato macrofagi hanno mostrato un aumento enzima lisosomiale ma una diminuzione uccidendo i microrganismi ingeriti 17. Tuttavia, la capacità dei fagociti non è stata influenzata se confrontato con i macrofagi non suscitato 16. Una volta coltivate in piatti, i macrofagi peritoneali diventano aderenti, consentendo quindi la loro separazione da altri tipi di cellule isolate dalla cavità peritoneale. Successivamente, i macrofagi isolati si sono sfidati con diversi agonisti TLR.Infine, mRNA è stato estratto dalle cellule in coltura e l&#39;espressione genica è stato analizzato utilizzando reazione inversa quantitativa a catena transcriptasi-polimerasi (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

Durante l&#39;infezione e l&#39;infiammazione, monociti circolanti lasciano il flusso sanguigno e migrare nei tessuti, dove si differenziano in macrofagi. I macrofagi esprimono i recettori di superficie Toll-like (TLR), che riconoscono pattern molecolari conservate attraverso l&#39;evoluzione in una vasta gamma di microrganismi. TLR svolgono un ruolo centrale nella attivazione dei macrofagi, che è di solito associata con l&#39;espressione genica alterazione. I macrofagi sono fondamentali per molte malattie e sono emersi come bersagli attraenti per la terapia. Nel seguente protocollo, si descrive una procedura per isolare i macrofagi peritoneali murini utilizzando medio Thioglycollate di birra. Quest&#39;ultimo incrementare la migrazione dei monociti nel peritoneo, di conseguenza tale da far salire il rendimento dei macrofagi per 10 volte. Diversi studi sono stati condotti utilizzando midollo osseo, milza o peritoneali derivati ​​da macrofagi. Tuttavia, i macrofagi peritoneali hanno dimostrato di essere più maturo sull&#39;isolamento e sono più stabili nella loro funzionalitàlità e fenotipo. Così, macrofagi isolati da murino cavità peritoneale presentano una popolazione importante cella che può servire in diversi studi immunologici e metabolici. Una volta isolato, i macrofagi sono state stimolate con differenti ligandi TLR e di conseguenza l&#39;espressione del gene è stata valutata.

Per prima cosa caratterizzati isolate macrofagi peritoneali murini mediante citometria a flusso. Per farlo, abbiamo utilizzato (F4 / 80) anticorpi che riconoscono specificamente i marcatori espressi solo da parte dei macrofagi. Questa caratterizzazione è necessaria per determinare la percentuale di isolati macrofagi e distinguerli tra cellule ottenute durante il processo di isolamento. Come mostrato in (figura 1), la percentuale di cellule che esprimono l&#39;antigene F4 / 80 è stata costantemente ris...

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.

Isolamento di murini peritoneale macrofagi di effettuare analisi di espressione genica Al Toll-like recettori stimolazione

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

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.

Isolamento di cellule della cavità peritoneale 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 ...

Ricerca

Immunologia e infezioni

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

Rilevazione di attività NEU1 sialidasi nella regolamentazione Toll-like l&#39;attivazione del recettore

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4 and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7. 
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-&kappa;B, which initiates transcription of numerous genes that are critical for immune responses8. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-&kappa;B pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9.

We describe use of ImageStream technology (<a href="http://www.amnis.com/" target="_blank">www.amnis.com</a>), which combines quantitative flow cytometry with simultaneous high-resolution digital imaging, to quantify cellular mechanisms of primary immune cells from well-defined patient cohorts. Our studies provide a blueprint for translational investigations to quantify lineage specific cellular responses in small samples from subject cohorts.

Imaging quantitativa di Lineage specifici Toll-like receptor-mediate Signaling in monociti e cellule dendritiche da piccoli campioni di sangue umano

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

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.

Isolamento, elaborazione ed analisi dei murini gengivali

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

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Uso di<em&gt; Shigella flexneri</em&gt; Per studiare le interazioni autofagia-Citoscheletro

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

L&#39;isolamento e l&#39;attivazione di linfociti murini

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila membrana esterna Vescicole: Isolamento e analisi del loro potenziale pro-infiammatori sui macrofagi

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento di Salmonella typhimurium-contenente fagosomi dai macrofagi

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

Utilizzando l'analisi filogenetica di indagare l'origine genica negli eucarioti

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

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.

Valutazione degli effetti di farmaci a base di anticorpi sul midollo osseo murino e l'attivazione dei macrofagi peritoneali

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

Isolamento di murini peritoneale macrofagi di effettuare analisi di espressione genica Al Toll-like recettori stimolazione

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