The Department of Transfusion Medicine, Clinical Center, National Institutes of Health, provided elutriated monocytes. This work was supported by the Intramural Research Program, National Heart, Lung, and Blood Institute, National Institutes of Health.

Leucaferesi è stata effettuata sotto protocollo di ricerca di un soggetto umano approvato da un National Institutes of Health revisione istituzionale bordo. 1. Isolamento e Crioconservazione dei monociti <ol><li> Ottenere cellule mononucleate da leucaferesi di donatori umani, e arricchire monociti dal continuo controcorrente centrifugazione dilavamento delle cellule mononucleari come descritto nei riferimenti 7,8. Ottenere circa 100 x 10 6 cellule elutriated (circa 80 - 90% monociti) in tampone elutriation specificato dal costruttore. Contare le cellule utilizzando un emocitometro. </li><li> monociti centrifuga in un tubo da 15 ml in polipropilene a 300 xg per 5 min a temperatura ambiente. </li><li> Rimuovere il surnatante e delicatamente risospendere le cellule in FBS seguite da dimetilsolfossido per ottenere una concentrazione finale di 90% FBS / 10% dimetilsolfossido e 50 x 10 6 cellule / ml. </li><li> Aggiungere ogni ml di sospensione cellulare to un individuo esageratamente. </li><li> Mettere i cryovials in un contenitore di congelamento delle cellule e trasferire ad un -80 ° C freezer per 24 ore prima del trasferimento a un serbatoio di stoccaggio esageratamente azoto liquido per la conservazione a lungo termine. </li></ol> 2. La differenziazione dei monociti in macrofagi <ol><li> Scongelare un esageratamente di cellule trasferendo rapidamente a bagnomaria C 37 ° e poi subito la rimozione quando la sospensione cellulare è stato scongelato circa il 70%. </li><li> Subito dopo completa scongelamento a temperatura ambiente, trasferire i 1 ml contenuti esageratamente in 50 ml di 37 ° C riscaldata Roswell Park Memorial Institute (RPMI) 1640 con 2 mM L-glutammina, 50 ng / ml M-CSF, 25 ng / ml interleuchina-10 (IL-10), e del 10% FBS (mezzo completo). </li><li> Trasferimento 25 ml di sospensione dei monociti in ciascuno dei due 75 cm 2 palloni di plastica di coltura cellulare. </li><li> Incubare culture in un 37 ° cultura incubatore di cellule C con 5% di CO 2/95% di aria per 48 ore. </li><li> Sciacquare il cultures 3 volte con 10 ml di RPMI 1640 medium (pre-riscaldato a 37 ° C), rimuovere delicatamente il terreno di coltura per evitare di spostare tutte le cellule debolmente collegate. </li><li> Dopo il risciacquo, aggiungere mezzo fresco completa, cambiando media ogni 2 giorni fino monociti differenziano e proliferare in misura sufficiente a diventare confluenti. Ciò richiede circa una settimana di coltura. </li></ol> 3. Raccolta macrofagi di avviare esperimenti <ol><li> Risciacquare macrofagi differenziati in boccetta 3 volte con 10 ml di pre-riscaldato soluzione salina tamponata con fosfato di 37 ° C Dulbecco senza Ca 2 + e Mg 2+ prima di aggiungere 10 ml preriscaldata acido etilendiamminotetraacetico tripsina-soluzione 37 ° C 0,25% (EDTA). </li><li> Posto pallone in coltura incubatore cellulare per 10 - 15 minuti momento in cui circa il 90% dei macrofagi dovuto arrotondati e distaccato. Verificare questo esame microscopico del pallone. </li><li> Aggiungere 10 ml di RMPI 1640 medium contenente il 10% FBS per stripsinizzazione superiore. </li><li> Trasferire la sospensione cellulare macrofagi dal pallone in un tubo da 50 ml polipropilene, centrifugare 300 xg per 5 min, e scartare il surnatante. </li><li> Risospendere delicatamente i macrofagi in 1 ml di terreno completo. </li><li> Mescolare 10 ml di sospensione cellulare macrofagi con 10 microlitri soluzione Trypan Blue e determinare il numero di cellule macrofagi e vitalità (tipicamente&gt; 95%) con un emocitometro. </li><li> Sulla base del numero di celle (di solito circa 15 - 18 x 10 5 macrofagi), aggiungere ulteriore mezzo completo per ottenere la densità cellulare di semina desiderata. Nota: 1 x 10 5 macrofagi in 1,5 ml di mezzo per pozzetto di una piastra 12 pozzetti produrrà una cultura quasi confluenti. </li><li> Dopo la semina, incubare macrofagi durante la notte in un 37 ° C incubatore di coltura cellulare con il 5% di CO 2/95% di aria per permettere l&#39;adesione cellulare. Poi, iniziare gli esperimenti desiderati. </li></ol>

<ol>
	<li>Dey, A., Allen, J., Hankey-Giblin, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+and+polarization+of+macrophages+in+inflammation:+blood+monocytes+versus+tissue+macrophages.">Ontogeny and polarization of macrophages in inflammation: blood monocytes versus tissue macrophages.</a> Front. Immunol. 5, 683 (2014).</li><li>Waldo, S. W., 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=Heterogeneity+of+human+macrophages+in+culture+and+in+atherosclerotic+plaques.">Heterogeneity of human macrophages in culture and in atherosclerotic plaques.</a> Am. J. Pathol. 172, 1112-1126 (2008).</li><li>Akagawa, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+heterogeneity+of+colony-stimulating+factor-induced+human+monocyte-derived+macrophages.">Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages.</a> Int. J. Hematol. 76, 27-34 (2002).</li><li>Akagawa, K. 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=Functional+heterogeneity+of+colony-stimulating+factor-induced+human+monocyte-derived+macrophages.">Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages.</a> Respirology. 11 Suppl, S32-S36 (2006).</li><li>Fleetwood, A. J., Lawrence, T., Hamilton, J. A., Cook, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-macrophage+colony-stimulating+factor+(CSF)+and+macrophage+CSF-dependent+macrophage+phenotypes+display+differences+in+cytokine+profiles+and+transcription+factor+activities:+implications+for+CSF+blockade+in+inflammation.">Granulocyte-macrophage colony-stimulating factor (CSF) and macrophage CSF-dependent macrophage phenotypes display differences in cytokine profiles and transcription factor activities: implications for CSF blockade in inflammation.</a> J. Immunol. 178, 5245-5252 (2007).</li><li>Lacey, D. C., 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=Defining+GM-CSF-+and+macrophage-CSF-dependent+macrophage+responses+by+in+vitro+models.">Defining GM-CSF- and macrophage-CSF-dependent macrophage responses by in vitro models.</a> J. Immunol. 188, 5752-5765 (2012).</li><li>Strasser, E. F., Eckstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+leukocyte+collection+and+monocyte+isolation+for+dendritic+cell+culture.">Optimization of leukocyte collection and monocyte isolation for dendritic cell culture.</a> Transfus. Med. Rev. 24, 130-139 (2010).</li><li>Kim, 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=Monocyte+enrichment+from+leukapheresis+products+by+using+the+Elutra+cell+separator.">Monocyte enrichment from leukapheresis products by using the Elutra cell separator.</a> Transfusion (Paris). 47, 2290-2296 (2007).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+exclusion+test+of+cell+viability.">Trypan blue exclusion test of cell viability.</a> Curr. Protoc. Immunol. Appendix 3 (Appendix 3B), (2001).</li><li>Anzinger, J. J., 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=Native+low-density+lipoprotein+uptake+by+macrophage+colony-stimulating+factor-differentiated+human+macrophages+is+mediated+by+macropinocytosis+and+micropinocytosis.">Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis.</a> Arterioscler. Thromb. Vasc. Biol. 30, 2022-2031 (2010).</li><li>Zhao, B., 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=Constitutive+receptor-independent+low+density+lipoprotein+uptake+and+cholesterol+accumulation+by+macrophages+differentiated+from+human+monocytes+with+macrophage-colony-stimulating+factor+(M-CSF).">Constitutive receptor-independent low density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M-CSF).</a> J. Biol. Chem. 281, 15757-15762 (2006).</li><li>Freeman, S. R., 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=ABCG1-mediated+generation+of+extracellular+cholesterol+microdomains.">ABCG1-mediated generation of extracellular cholesterol microdomains.</a> J. Lipid Res. 55, 115-127 (2014).</li><li>Kruth, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor-independent+fluid-phase+pinocytosis+mechanisms+for+induction+of+foam+cell+formation+with+native+low-density+lipoprotein+particles.">Receptor-independent fluid-phase pinocytosis mechanisms for induction of foam cell formation with native low-density lipoprotein particles.</a> Curr. Opin. Lipidol. 22, 386-393 (2011).</li><li>Jin, X., 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=ABCA1+contributes+to+macrophage+deposition+of+extracellular+cholesterol.">ABCA1 contributes to macrophage deposition of extracellular cholesterol.</a> J. Lipid Res. 56, 1720-1726 (2015).</li><li>Ong, D. 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=Extracellular+cholesterol-rich+microdomains+generated+by+human+macrophages+and+their+potential+function+in+reverse+cholesterol+transport.">Extracellular cholesterol-rich microdomains generated by human macrophages and their potential function in reverse cholesterol transport.</a> J. Lipid Res. 51, 2303-2313 (2010).</li><li>Hashimoto, S., Yamada, M., Motoyoshi, K., Akagawa, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+macrophage+colony-stimulating+factor-induced+growth+and+differentiation+of+human+monocytes+by+interleukin-10.">Enhancement of macrophage colony-stimulating factor-induced growth and differentiation of human monocytes by interleukin-10.</a> Blood. 89, 315-321 (1997).</li><li>Hashimoto, S. I., Komuro, I., Yamada, M., Akagawa, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-10+inhibits+granulocyte-macrophage+colony-stimulating+factor-dependent+human+monocyte+survival+at+the+early+stage+of+the+culture+and+inhibits+the+generation+of+macrophages.">IL-10 inhibits granulocyte-macrophage colony-stimulating factor-dependent human monocyte survival at the early stage of the culture and inhibits the generation of macrophages.</a> J. Immunol. 167, 3619-3625 (2001).</li><li>Lund, P. K., Joo, G. B., Westvik, A. B., Ovstebo, R., Kierulf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+monocytes+from+whole+blood+by+density+gradient+centrifugation+and+counter-current+elutriation+followed+by+cryopreservation:+six+years'+experience.">Isolation of monocytes from whole blood by density gradient centrifugation and counter-current elutriation followed by cryopreservation: six years' experience.</a> Scand. J. Clin. Lab. Invest. 60, 357-365 (2000).</li><li>Weiner, R. S., Normann, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+integrity+of+cryopreserved+human+monocytes.">Functional integrity of cryopreserved human monocytes.</a> J. Natl. Cancer Inst. 66, 255-260 (1981).</li><li>Hansen, J. B., 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=Retention+of+phagocytic+functions+in+cryopreserved+human+monocytes.">Retention of phagocytic functions in cryopreserved human monocytes.</a> J. Leukoc. Biol. 57, 235-241 (1995).</li><li>Seager Danciger, J., 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=Method+for+large+scale+isolation,+culture+and+cryopreservation+of+human+monocytes+suitable+for+chemotaxis,+cellular+adhesion+assays,+macrophage+and+dendritic+cell+differentiation.">Method for large scale isolation, culture and cryopreservation of human monocytes suitable for chemotaxis, cellular adhesion assays, macrophage and dendritic cell differentiation.</a> J. Immunol. Methods. 288, 123-134 (2004).</li><li>Jin, X., Xu, Q., Champion, K., Kruth, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin+contamination+of+apolipoprotein+A-I:+effect+on+macrophage+proliferation--a+cautionary+tale.">Endotoxin contamination of apolipoprotein A-I: effect on macrophage proliferation--a cautionary tale.</a> Atherosclerosis. 240, 121-124 (2015).</li></ol>

The authors have nothing to disclose.

Generazione di tipi definiti macrofagi può chiarire alcuni dei risultati contrastanti ottenuti dai ricercatori nello studio della biologia dei macrofagi. L&#39;uso di varie condizioni di coltura e fattori di differenziazione per generare macrofagi umani primari possono portare a tipi molto diversi macrofagi, un fatto che non può essere apprezzata dal ricercatore. Ad esempio, i macrofagi a volte sono generate da monociti umani non usando il siero, siero umano da solo, siero umano integrato con M-CSF, FBS da solo, o FBS contenenti M-CSF o GM-CSF. Come riportato in precedenza e come abbiamo anche osservato, monociti in coltura per una settimana senza siero o con FBS senza fattori di differenziazione aggiunti non rimanere vitale 3. Come accennato in precedenza, monociti in coltura con siero umano da solo o integrato con M-CSF generare macrofagi che mostrano il &quot;uovo fritto&quot; morfologia caratteristica dei monociti differenziati con FBS inclusa GM-CSF. Pertanto, in presenza di siero umano M-Tipo CSF ​​macrofagi che mostra una morfologia più allungata, non si ottiene anche se monociti sono stati differenziati con M-CSF. È interessante notare, abbiamo osservato che la differenziazione dei monociti con FBS contenenti sia M-CSF e GM-CSF, genera sia l&#39;M-CSF allungata e GM-CSF &quot;uovo fritto&quot; fenotipi morfologici nella stessa cultura. Questo suggerisce che distinte sottopopolazioni monociti danno origine a questi due tipi di macrofagi. Studi precedenti hanno dimostrato che M-CSF e fattori di differenziazione GM-CSF influenzano non solo la morfologia del macrofago, ma anche influenzare l&#39;espressione genica e la funzione delle cellule, che è sostanzialmente differente per questi due tipi macrofagi 2-6. Immunocolorazione per il CD68 marcatore macrofagi in collaborazione con CD14 che si esprime per tipo macrofagi M-CSF e 25F9 che si esprime da parte dei macrofagi GM-CSF può essere utilizzata per confermare il tipo di macrofagi, come indicato in precedenza 2. Questi due tipi di macrofagi may essere particolarmente rilevante per lo studio dell&#39;aterosclerosi, dove diversi tipi di macrofagi si verificano in placche aterosclerotiche e possono mostrare differenze nel metabolismo del colesterolo e di espressione dei mediatori dell&#39;infiammazione 2,12,13. Ad esempio, digitare M-CSF macrofagi sono unici in quanto questi macrofagi deposito di colesterolo nella matrice extracellulare, quando i macrofagi sono arricchiti con il colesterolo 12,14,15. Nel protocollo presentato, durante la coltura con M-CSF, IL-10 è stato aggiunto ordinariamente come descritto in precedenza 16. Questa citochina promuove la crescita e la differenziazione dei macrofagi e produce una morfologia allungata più omogenea dei macrofagi. Inoltre, IL-10 inibisce la sopravvivenza monociti GM-CSF-dipendente inibendo gli eventi di segnalazione indotti da GM-CSF 17. Tuttavia, gli investigatori dovrebbero considerare che l&#39;IL-10 può influenzare le altre risposte delle cellule in fase di studio, e di conseguenza, potrebbe voler omettere IL-10. Primautilizzando il protocollo corrente utilizzando la proliferazione e la differenziazione dei monociti in un pallone, culture macrofagi sono state avviate dalla placcatura direttamente monociti elutriated nei pozzetti cultura e differenziandole per 7 giorni prima di iniziare gli esperimenti. Tuttavia, anche con l&#39;aggiunta di IL-10, alcune di queste culture di macrofagi mostrato piccole quantità (di solito meno di 10%) di &quot;uovo fritto&quot; tipo GM-CSF macrofagi contaminanti le allungati M-CSF tipo macrofagi. Con l&#39;attuale protocollo, GM-CSF contaminazione tipo di macrofagi non si verifica per ragioni che non sono chiare. Tuttavia, non è perché GM-CSF di tipo macrofago monociti precursori sono persi durante il congelamento, come coltura i monociti crioconservati con FBS + GM-CSF produce eccellenti culture di tipo GM-CSF (vedere Figura 4). Il protocollo descritto utilizza la crioconservazione dei monociti prima della loro differenziazione in macrofagi. Monociti crioconservazione è stato previously dimostrato di non influire sulla funzione dei monociti e viene comunemente impiegato 18-21. A questo proposito, abbiamo trovato che i macrofagi generati dal protocollo sopra colesterolo deposito nello spazio extracellulare simile a quanto descritto in precedenza con monociti non crioconservati che sono state coltivate direttamente in macrofagi 12. Con monociti crioconservazione, monociti sono sempre disponibili per avviare esperimenti indipendenti di quando i monociti possono essere ottenute da un donatore. Anche se non è essenziale per crioconservare i monociti prima di procedere con il protocollo, la crioconservazione ha prodotto culture macrofagi più omogenee in quanto tutti i macrofagi erano più diffuso, piuttosto che essere sia arrotondata e diffondere come era il caso in cui monociti freschi sono stati usati per avviare culture. Abbiamo generato con successo culture macrofagi da monociti che sono stati mantenuti congelati fino a 6 mesi, (il più lungo tempo testato). D&#39;altra parte, la semina di MACR crioconservatiophages nella cultura pozzi per gli esperimenti non hanno prodotto adeguate culture macrofagi. passaggi critici nel protocollo comprendono immediatamente mettendo monociti nel congelatore in seguito all&#39;aggiunta di dimetilsolfossido, e subito il trasferimento di monociti scongelati al mezzo di coltura. Anche se è comune per rimuovere DMSO mediante centrifugazione cellule congelate prima di essere seminate nella cultura, non abbiamo trovato questo era un passo necessario nella cultura di successo dei monociti. Tuttavia, gli investigatori possono avere bisogno di fare questo se la concentrazione di DMSO residua (2 ml / ml) è superiore a quello che è stato utilizzato nel protocollo di cui sopra. E &#39;importante non lavare i monociti fino a 48 ore dopo la loro semina in fiasche di coltura. Altrimenti, alcuni monociti possono essere persi a seconda del loro grado di adesione alla superficie cultura. D&#39;altra parte, una volta che i monociti differenziano in macrofagi, aumentata adesione dei macrofagi può richiedere più tripsina trattamento per rimuoverli,o un sollevatore cella può essere impiegato per facilitare stampa dopo il trattamento iniziale tripsina. Se monociti non proliferano e si differenziano, questo potrebbe essere causa di alcuni insoliti caratteristiche del donatore monociti, perdita di attività di M-CSF, o per il lotto di FBS utilizzato. Inoltre, di possibile preoccupazione quando le culture non si sviluppa è la contaminazione dei reagenti con endotossine, un agente che inibisce la proliferazione dei macrofagi 22. Differenziare i monociti crioconservati in boccette con M-CSF permette loro differenziazione di massa in macrofagi. Poi, i macrofagi possono essere raccolte e seminate in pozzetti di coltura a densità di cellule necessarie per lo svolgimento di esperimenti. L&#39;uso dei numeri definiti di macrofagi piuttosto che numeri definite di monociti di avviare colture di macrofagi per esperimenti, produce colture di macrofagi in cui la densità cellulare desiderato può essere raggiunto più coerente.

Study of cultured macrophages is a useful model to understand the function of these cells in inflammation such as occurs in atherosclerotic plaques. When the research focus is on human diseases involving macrophages, it is useful to study primary human macrophages rather than non-human macrophages to avoid species differences. Also, the effects of cell transformation can be avoided by using primary macrophages rather than macrophage cell lines. For this purpose, macrophages differentiated from monocytes isolated from human blood serve as a means of obtaining primary human macrophages. 
Tissue macrophages may be either resident within tissues or may be derived from monocytes that migrate into the tissue and differentiate into macrophages 1. Two types of human monocyte-derived macrophages have been defined that differ not only in morphology but also gene expression and cell function 2. These two types are obtained from monocytes that are differentiated into macrophages in the presence of M-CSF + fetal bovine serum (FBS) and monocytes that are differentiated into macrophages in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) + FBS 2-6. In our experience (unpublished observation), use of human serum rather than FBS generates the GM-CSF type regardless of whether M-CSF or GM-CSF is included in the differentiation medium. M-CSF type macrophages tend to be more elongated than GM-CSF type macrophages, which resemble fried eggs in their morphology. Investigators should recognize that human M-CSF and GM-CSF monocyte-derived macrophages are not the same as so called M1 and M2 mouse bone marrow-derived macrophages 6.
During research using human monocyte-derived M-CSF differentiated macrophages, we experienced difficulty related to the availability of monocytes to initiate experiments and variation in the obtained cell densities of macrophages during differentiation of monocytes into macrophages. To overcome these problems, we have developed the following protocol in which monocytes are frozen until required for use, and monocytes are differentiated in bulk into macrophages that can then be removed from culture flasks and plated at desired cell densities to obtain more uniform cultures from experiment to experiment.

<table border="0" cellpadding="0" cellspacing="0" width="1086">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Cellbind 12-well culture plate</td>
			<td style="width:319px;">Corning</td>
			<td style="width:173px;">3336</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">CELLSTAR, T-75 flask, tissue culture treated</td>
			<td style="width:319px;">Greiner Bio-One North America</td>
			<td style="width:173px;">658157</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">RPMI 1640 culture medium</td>
			<td style="width:319px;">Cellgro Mediatech</td>
			<td style="width:173px;">15-040-CM</td>
			<td style="width:167px;">warmed to 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">L-Glutamine</td>
			<td style="width:319px;">Cellgro Mediatech</td>
			<td style="width:173px;">25-005-CI</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Fetal bovine serum</td>
			<td style="width:319px;">Gibco</td>
			<td style="width:173px;">16000-036</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">M-CSF</td>
			<td style="width:319px;">PeproTech</td>
			<td style="width:173px;">300-25</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">GM-CSF</td>
			<td style="width:319px;">PeproTech</td>
			<td style="width:173px;">300-03</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">IL-10&#160;</td>
			<td style="width:319px;">PeproTech</td>
			<td style="width:173px;">200-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">DMSO</td>
			<td style="width:319px;">Sigma</td>
			<td style="width:173px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Cryovial&#160;</td>
			<td style="width:319px;">Thermo Scientific&#160;</td>
			<td style="width:173px;">375418</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:428px;">DPBS without Ca2+ and Mg2+&#160;</td>
			<td style="width:319px;">Corning cellgro</td>
			<td style="width:173px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">0.25% Trypsin-EDTA&#160;</td>
			<td style="width:319px;">Gibco</td>
			<td style="width:173px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">50 ml polypropylene conical tube</td>
			<td style="width:319px;">Falcon</td>
			<td style="width:173px;">352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Trypan Blue</td>
			<td style="width:319px;">Lonza</td>
			<td style="width:173px;">17-942E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Neubauer-improved bright light hemocytometer&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:319px;">Paul Marienfeld GmbH &#38; Co. KG</td>
			<td style="width:173px;">610031</td>
			<td>http://www.marienfeld-superior.com/index.php/counting-chambers/articles/counting-chambers.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">CoolCell LX cell freezing container</td>
			<td style="width:319px;">BioCision</td>
			<td style="width:173px;">BCS-405</td>
			<td>other freezing containers also should&#160; be adequate for this step</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Liquid Nitrogen Storage System, CryoPlus 1</td>
			<td style="width:319px;">Thermo Scientific&#160;</td>
			<td style="width:173px;">7400</td>
			<td>any liquid nitrogen tank should be adequate</td>
		</tr>
	</tbody>
</table>

culture of macrophage colony-stimulating factor differentiated human monocyte-derived macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

La vitalità dei monociti freschi o crioconservati era superiore al 95%, come determinato con Trypan Blue colorazione 9. Figura 1 e Figura 2 confronta ad ingrandimenti inferiori e superiori, rispettivamente, l&#39;andamento di fresco e crioconservato (cioè, congelato) differenziazione dei monociti in macrofagi. Si noti che il fresco confrontato con monociti crioconservati mostrano una sottopopolazione di differenziare i monociti che non si diffondono, ma restano arrotondato. vacuoli Phase-Lucent verificano nei macrofagi derivate da monociti in entrambe le condizioni. Questi vacuoli rappresentano caratteristica macropinosomes di tipo macrofagi M-CSF come descritto in precedenza 10,11. macrofagi Cryopreserving dopo la loro raccolta dai contenitori in cui sono stati differenziati da monociti non hanno prodotto culture macrofagi di successo ( <sTrong&gt; Figura 3). Tali culture hanno mostrato attaccamento cellulare molto scarsa probabilmente a causa della scarsa redditività dopo crioconservazione. Il protocollo di cui sopra per la produzione di tipo M-CSF macrofagi fa senza la presenza di contaminanti di tipo GM-CSF macrofagi che a volte si verifica quando le culture sono stabiliti direttamente da monociti senza il loro crioconservazione e la differenziazione di tipo M-CSF macrofagi in fiaschi. Tuttavia, monociti crioconservati differenziati con FBS + GM-CSF produce eccellenti culture di tipo GM-CSF (Figura 4). <img alt="Figura 1" src="/files/ftp_upload/54244/54244fig1.jpg" /> Figura 1: Confronto di fresco contro monociti crioconservati utilizzato per la produzione di macrofagi (basso ingrandimento) a contrasto di fase immagini microscopiche di monociti crioconservati (congelati) e fresche durante la loro M-CSF indotta differenziazione i.<html> macrofagi nto. vengono riportati i monociti durante la coltura in fiasche a 3 e 7 giorni, e il giorno 8, un giorno dopo la loro raccolta e replating in piastre di coltura da 12 pozzetti. Bar = 100 micron e si applica a tutti. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54244/54244fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/54244/54244fig2.jpg" /> Figura 2:. Confronto di fresco contro monociti crioconservati utilizzati per la produzione di macrofagi (alto ingrandimento) I monociti sono stati coltivati ​​come descritto nella Figura 1 leggenda e mostrato qui a maggiore ingrandimento. vacuoli fase-Lucent sono macropinosomes (frecce bianche). Bar = 50 micron e si applica a tutti. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54244/54244fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> tenda &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; <img alt="Figura 3" src="/files/ftp_upload/54244/54244fig3.jpg" /> Figura 3: crioconservati macrofagi non riescono a produrre colture adeguate monociti fresche sono state seminate in un pallone e differenziati 7 giorni con M-CSF come mostrato nelle figure 1 e 2.. Poi, i macrofagi differenziati sono state raccolte dai fiaschi e crioconservati. Successivamente, i macrofagi crioconservati sono state seminate in piastre da 12 pozzetti come descritto nel protocollo per macrofagi non crioconservati e coltivate 1 giorno (giorno 8). A contrasto di fase immagine microscopica della cultura 1 giorno dimostra che ben pochi dei macrofagi attach dopo la loro crioconservazione (confrontare in giorno 8 risultati per i macrofagi non crioconservati in figura 1 di cui sopra). Bar = 100 micron. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54244/54244fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> Figura 4:. Monociti crioconservati erano anche adatto a produrre tipo GM-CSF macrofagi derivate da monociti monociti crioconservati (25 x 10 6) sono state seminate in un pallone e differenziati 8 giorni con 10% FBS contenente 50 ng / ml di GM-CSF. immagini a contrasto di fase mostra la morfologia tipica &quot;uovo fritto&quot; di tipo macrofagi GM-CSF. Bar = 100 micron. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54244/54244fig4large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages at JoVE.com

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Cultura dei macrofagi macrofagi colony-stimulating factor differenziata umana derivate da monociti

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

culture-macrophage-colony-stimulating-factor-differentiated-human

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

29.0K Views.  National Institutes of Health. A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Video: Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Un<em&gt; In vitro</em&gt; Co-infezione da modello per lo studio<em&gt; Plasmodium falciparum</em&gt;-HIV-1 in Interazioni umane Le cellule immunitarie primarie derivate da monociti

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Ricerca

Immunologia e infezioni

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Indagine di polarizzazione dei macrofagi Utilizzando Bone Marrow macrofagi derivati

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Attivazione e misurazione dei NLRP3 inflammasome all&#39;attività utilizzando IL-1β in cellule dendritiche umane derivate da monociti

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Caratterizzazione delle derivate da monociti cellule dendritiche umane di Citometria a Flusso Imaging: un confronto tra due protocolli di isolamento dei monociti

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generazione di derivate da monociti cellule dendritiche umane da sangue intero

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Analisi dei Macrofagi Microglia e dei Monociti dal Sistema Nervoso Centrale dalla Citometria del Flusso

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolamento e coltura In Vitro dei macrofagi alveolari murini ed umani

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

In Vitro Differenziazione dei Mouse del Granulocyte-macrofago-colony-stimulating Factor (GM-CSF)-producendo le cellule T Helper (THGM)

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

Quantificazione dell'attività chemiotatica monocite in vivo e caratterizzazione dei macrofagi derivati monociti del sangue

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

Stimolazione in vitro e visualizzazione del rilascio di trap extracellulari nei Macrofagi derivati da Monciti Umani differenziati

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...