Name: generation and identification of gm-csf derived alveolar-like macrophages and dendritic cells from mouse bone marrow
Uploaded: 2016-06-25T14:00:00
Description: Watch this Scientific Journal Video about Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow at JoVE.com

इस काम के स्वास्थ्य अनुसंधान (CIHR) (ग्रांट एमओपी 119,503) और कनाडा के प्राकृतिक विज्ञान और इंजीनियरिंग परिषद (NSERC) की कनाडा के संस्थानों द्वारा वित्त पोषित किया गया था। NSERC भी गर्मियों studentships समर्थित यार्ड के लिए और ए.ए. वाई.डी. ब्रिटिश कोलंबिया विश्वविद्यालय (यूबीसी) एक 4 साल फैलोशिप पुरस्कार के साथ द्वारा समर्थित है, ए.ए. एक स्नातक छात्र मास्टर पुरस्कार (सीजीएस-एम) के साथ CIHR द्वारा समर्थित है। हम चित्रा 2 में छवियों को उत्पन्न करने के लिए इस्तेमाल माइक्रोस्कोप के साथ सहायता के लिए केल्विन Roskelley धन्यवाद। हम यह भी यूबीसी पशु और से फ्लो सुविधाओं से समर्थन स्वीकार करते हैं।

चूहे ब्रिटिश कोलंबिया पशु की देखभाल समिति के विश्वविद्यालय द्वारा अनुमोदित प्रक्रियाओं द्वारा नैतिक पशु अनुसंधान के लिए पशु की देखभाल के दिशा-निर्देशों पर कनाडा परिषद के अनुसार euthanized थे। 1. माउस फीम...

<ol>
	<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 (8), 5245-5252 (2007).</li><li>Lari, 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=Macrophage+lineage+phenotypes+and+osteoclastogenesis--complexity+in+the+control+by+GM-CSF+and.">Macrophage lineage phenotypes and osteoclastogenesis--complexity in the control by GM-CSF and.</a> Bone. 40 (2), 323-336 (2007).</li><li>Brasel, K., De Smedt, T., Smith, J. L., Maliszewski, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+murine+dendritic+cells+from+flt3-ligand-supplemented+bone+marrow+cultures.">Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures.</a> Blood. 96 (9), 3029-3039 (2000).</li><li>Angelov, G. S., Tomkowiak, M., Marcais, A., Leverrier, Y., Marvel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flt3+ligand-generated+murine+plasmacytoid+and+conventional+dendritic+cells+differ+in+their+capacity+to+prime+naive+CD8+T+cells+and+to+generate+memory+cells+in+vivo.">Flt3 ligand-generated murine plasmacytoid and conventional dendritic cells differ in their capacity to prime naive CD8 T cells and to generate memory cells in vivo.</a> J Immunol. 175 (1), 189-195 (2005).</li><li>Inaba, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+large+numbers+of+dendritic+cells+from+mouse+bone+marrow+cultures+supplemented+with+granulocyte/macrophage+colony-stimulating+factor.">Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.</a> J Exp Med. 176 (6), 1693-1702 (1992).</li><li>Lutz, M. 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=An+advanced+culture+method+for+generating+large+quantities+of+highly+pure+dendritic+cells+from+mouse+bone+marrow.">An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow.</a> J Immunol Methods. 223 (1), 77-92 (1999).</li><li>Labeur, M. 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=Generation+of+tumor+immunity+by+bone+marrow-derived+dendritic+cells+correlates+with+dendritic+cell+maturation+stage.">Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage.</a> J Immunol. 162 (1), 168-175 (1999).</li><li>Berthier, R., Martinon-Ego, C., Laharie, A. M., Marche, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-step+culture+method+starting+with+early+growth+factors+permits+enhanced+production+of+functional+dendritic+cells+from+murine+splenocytes.">A two-step culture method starting with early growth factors permits enhanced production of functional dendritic cells from murine splenocytes.</a> J Immunol Methods. 239 (1-2), 95-107 (2000).</li><li>Brasel, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flt3+ligand+synergizes+with+granulocyte-macrophage+colony-stimulating+factor+or+granulocyte+colony-stimulating+factor+to+mobilize+hematopoietic+progenitor+cells+into+the+peripheral+blood+of+mice.">Flt3 ligand synergizes with granulocyte-macrophage colony-stimulating factor or granulocyte colony-stimulating factor to mobilize hematopoietic progenitor cells into the peripheral blood of mice.</a> Blood. 90 (9), 3781-3788 (1997).</li><li>Segura, E., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+dendritic+cells+in+mice+and+humans.">Inflammatory dendritic cells in mice and humans.</a> Trends Immunol. 34 (9), 440-445 (2013).</li><li>West, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+dendritic+cell+antigen+capture+via+toll-like+receptor-induced+actin+remodeling.">Enhanced dendritic cell antigen capture via toll-like receptor-induced actin remodeling.</a> Science. 305 (5687), 1153-1157 (2004).</li><li>Veldhoen, M., Hocking, R. J., Atkins, C. J., Locksley, R. M., Stockinger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta+in+the+context+of+an+inflammatory+cytokine+milieu+supports+de+novo+differentiation+of+IL-17-producing+T+cells.">TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells.</a> Immunity. 24 (2), 179-189 (2006).</li><li>Goodridge, H. 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=Activation+of+the+innate+immune+receptor+Dectin-1+upon+formation+of+a+'phagocytic+synapse.">Activation of the innate immune receptor Dectin-1 upon formation of a 'phagocytic synapse.</a> Nature. 472 (7344), 471-475 (2011).</li><li>Shalek, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA-seq+reveals+dynamic+paracrine+control+of+cellular+variation.">Single-cell RNA-seq reveals dynamic paracrine control of cellular variation.</a> Nature. 510 (7505), 363-369 (2014).</li><li>Vander Lugt, 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=Transcriptional+programming+of+dendritic+cells+for+enhanced+MHC+class+II+antigen+presentation.">Transcriptional programming of dendritic cells for enhanced MHC class II antigen presentation.</a> Nat Immunol. 15 (2), 161-167 (2014).</li><li>Shibata, Y., 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=GM-CSF+regulates+alveolar+macrophage+differentiation+and+innate+immunity+in+the+lung+through+PU.1.">GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU.1.</a> Immunity. 15 (4), 557-567 (2001).</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 (11), 5752-5765 (2012).</li><li>Poon, G. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan+Binding+Identifies+a+Functionally+Distinct+Alveolar+Macrophage-like+Population+in+Bone+Marrow-Derived+Dendritic+Cell+Cultures.">Hyaluronan Binding Identifies a Functionally Distinct Alveolar Macrophage-like Population in Bone Marrow-Derived Dendritic Cell Cultures.</a> J Immunol. 195 (2), 632-642 (2015).</li><li>Helft, 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=GM-CSF+Mouse+Bone+Marrow+Cultures+Comprise+a+Heterogeneous+Population+of+CD11c(+)MHCII(+)+Macrophages+and+Dendritic+Cells.">GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous Population of CD11c(+)MHCII(+) Macrophages and Dendritic Cells.</a> Immunity. 42 (6), 1197-1211 (2015).</li><li>Stockinger, B., Zal, T., Zal, A., Gray, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cells+solicit+their+own+help+from+T+cells.">cells solicit their own help from T cells.</a> J. Exp. Med. 183 (3), 891-899 (1996).</li><li>de Belder, A. N., Wik, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+properties+of+fluorescein-labelled+hyaluronate.">Preparation and properties of fluorescein-labelled hyaluronate.</a> Carbohydr Res. 44 (2), 251-257 (1975).</li><li>Stewart, C. C., Stewart, 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=Immunophenotyping.">Immunophenotyping.</a> Current Protocols in Cytometry. , (2001).</li><li>Dearman, R. J., Cumberbatch, M., Maxwell, G., Basketter, D. A., Kimber, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+ligand+activation+of+murine+bone+marrow-derived+dendritic+cells.">Toll-like receptor ligand activation of murine bone marrow-derived dendritic cells.</a> Immunology. 126 (4), 475-484 (2009).</li><li>Abdi, K., Singh, N. J., Matzinger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-Activated+Dendritic+Cells:+'Exhausted'+or+Alert+and+Waiting.">Lipopolysaccharide-Activated Dendritic Cells: 'Exhausted' or Alert and Waiting.</a> J Immunol. 188 (12), 5981-5989 (2012).</li><li>Contreras, I., 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=Impact+of+Leishmania+mexicana+Infection+on+Dendritic+Cell+Signaling+and+Functions.">Impact of Leishmania mexicana Infection on Dendritic Cell Signaling and Functions.</a> PLoS Negl Trop Dis. 8 (9), (2014).</li><li>Feng, T., Cong, Y. Z., Qin, H. W., Benveniste, E. N., Elson, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Mucosal+Dendritic+Cells+from+Bone+Marrow+Reveals+a+Critical+Role+of+Retinoic+Acid.">Generation of Mucosal Dendritic Cells from Bone Marrow Reveals a Critical Role of Retinoic Acid.</a> J Immunol. 185 (10), 5915-5925 (2010).</li><li>Grauer, O., 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=Analysis+of+maturation+states+of+rat+bone+marrow-derived+dendritic+cells+using+an+improved+culture+technique.">Analysis of maturation states of rat bone marrow-derived dendritic cells using an improved culture technique.</a> Histochem Cell Biol. 117 (4), 351-362 (2002).</li><li>Suzuki, T., 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=Pulmonary+macrophage+transplantation+therapy.">Pulmonary macrophage transplantation therapy.</a> Nature. 514 (7523), 450-454 (2014).</li><li>Happle, 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=Pulmonary+transplantation+of+macrophage+progenitors+as+effective+and+long-lasting+therapy+for+hereditary+pulmonary+alveolar+proteinosis.">Pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for hereditary pulmonary alveolar proteinosis.</a> Sci Transl Med. 6 (250), (2014).</li></ol>

इस पांडुलिपि में, हम एक माउस अस्थि मज्जा संस्कृति है कि लुट्ज़ एट अल। 6 से अनुकूलित है से जीएम- CSF व्युत्पन्न मैक्रोफेज और DCS पैदा करने के लिए एक विधि प्रदान करते हैं। MHCII अभिव्यक्ति और फ्लोरिडा हा इ?...

कई इन विट्रो संस्कृति तरीकों अस्थि मज्जा व्युत्पन्न मैक्रोफेज (BMDMs) और अस्थि मज्जा व्युत्पन्न DCS (BMDCs) एक या विकास के कारकों के संयोजन का उपयोग उत्पन्न करने के लिए वर्णित किया गया है। BMDMs या तो बृहतभक्षककोशिका कॉलोनी उत्तेजक कारक (एम-सीएसएफ) या जीएम- CSF 1,2 का उपयोग कर अस्थि मज्जा की कोशिकाओं संवर्धन द्वारा उत्पन्न किया जा सकता है। BMDCs के लिए, अस्थि मज्जा संस्कृति को FLT3 ligand के अलावा गैर पक्षपाती शास्त्रीय और plasmacytoid डीसी को जन्म (CD11c उच्च / MHCII उच्च और CD11c लो, B220 + क्रमशः) देता संस्कृति 3,4 9 दिनों के बाद। इसके विपरीत, गैर पक्षपाती कोशिकाओं अकेले जीएम- CSF 5,6 के साथ संस्कृति में 7 से 10 दिनों के बाद उत्पन्न, जीएम-सीएसएफ और आईएल 4 से 7, या जीएम- CSF और FLT3 ligand 8,9 उत्पन्न BMDCs अधिक बारीकी से भड़काऊ डीसी जैसी (CD11c उच्च, MHCII उच्च CD11b +) 10। इन विट्रो में संस्कृतियों मैक्रोफेज उत्पन्न करने के लिए उपयोग किया जाता है याडीसी, यह स्पष्ट नहीं है, तो प्रत्येक संस्कृति शुद्ध आबादी को जन्म देता है। उदाहरण के लिए, हालांकि जीएम- CSF संस्कृतियों में पक्षपाती कोशिकाओं मैक्रोफेज 5, एक ही संस्कृति से गैर पक्षपाती कोशिकाओं डीसी 6,11-13 के रूप में उपयोग किया जाता है, अनुमान है कि वे सजातीय हैं और किसी भी मनाया परिवर्तनशीलता है साथ होने के लिए वर्णित हैं कारण विकास 14,15 के विभिन्न चरणों के लिए। इसके अलावा, अध्ययन विवो 16,17 में वायुकोशीय मैक्रोफेज के विकास के लिए एक आवश्यक विकास कारक हो जीएम- CSF पाया है, और वायुकोशीय तरह मैक्रोफेज 16,17,18 उत्पन्न करने के लिए इन विट्रो में इस्तेमाल किया जा सकता है। पालन ​​के अलावा, जीएम- CSF इलाज किया अस्थि मज्जा संस्कृतियों से मैक्रोफेज और DCS पैदा करने के लिए प्रक्रिया बहुत विविधता का सुझाव जीएम- CSF अस्थि मज्जा संस्कृतियों के भीतर मौजूद हो सकता है के समान हैं। यह वास्तव में के रूप में दो पत्र BMDC संस्कृतियों की गैर पक्षपाती अंश में BMDMs की उपस्थिति की रिपोर्ट का मामला प्रतीत हो रहा है। एक पत्र में, वे identifiप्रवर्तन निदेशालय के रूप CD11c +, CD11b +, MHCII मध्य, MerTK +, और CD115 +, जो एक जीन की अभिव्यक्ति के हस्ताक्षर है कि सबसे निकट वायुकोशीय मैक्रोफेज मची और टी कोशिकाओं 19 को सक्रिय करने के लिए एक कम क्षमता थी व्यक्त कोशिकाओं की आबादी। (CD11c +, MHCII मध्य / कम, फ्लोरिडा हा उच्च) कि अपरिपक्व से अलग किया गया था (CD11c +, MHCII मध्य, फ्लोरिडा हा कम) और परिपक्व दूसरे का इस्तेमाल किया MHCII और फ्लोरिडा हा कागज एक वायुकोशीय बृहतभक्षककोशिका की तरह जनसंख्या की पहचान करने के लिए DCS (CD11c +, MHCII उच्च), दोनों phenotypically और कार्यात्मक 18। इन पत्रों में दोनों उदाहरण देकर स्पष्ट करना है कि जीएम-सीएसएफ BMDC संस्कृतियों विषम हैं, दोनों मैक्रोफेज और उस देखभाल का संकेत है जब BMDC संस्कृतियों से डेटा की व्याख्या लिया जाना चाहिए डीसी आबादी से युक्त। इस प्रोटोकॉल का वर्णन अस्थि मज्जा, जीएम- CSF में संस्कृति अस्थि मज्जा की कोशिकाओं को अलग-थलग करने के लिए कैसे, और वायुकोशीय बृहतभक्षककोशिका तरह की पहचानअपरिपक्व और परिपक्व बंधन और MHCII अभिव्यक्ति FL-हा का उपयोग कर प्रवाह cytometry द्वारा अस्थि मज्जा संस्कृति में डीसी से आबादी। । यह प्रक्रिया लुट्ज़ एट अल 6 की स्थापना की प्रक्रिया पर आधारित है और 5 उत्पन्न करने में सक्षम है - 10 x 10 6 गैर पक्षपाती एक 10 मिलीलीटर संस्कृति के 7 दिन पर कोशिकाओं। संस्कृति दिनों से 7 से 10 प्रयोग करने योग्य है और 7. यह आगे बढ़ने और बड़ी मात्रा में अलग-थलग इन विट्रो वायुकोशीय तरह मैक्रोफेज के लिए एक सरल तरीका प्रदान करता है दिन में मैक्रोफेज, अपरिपक्व और परिपक्व DCs, साथ ही कुछ progenitors के एक विषम आबादी पैदावार।

<table>
	<tbody>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD&#160;</td>
			<td>LSR-II</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Inverted Microscope&#160;</td>
			<td>Leica&#160;</td>
			<td>DMI4000 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Thermo Fisher</td>
			<td>ST-40R</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinet</td>
			<td>Nuaire</td>
			<td>NU-425-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1 ml</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>26 1/2 Gauge Needle</td>
			<td>BD</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Conical Tube&#160;</td>
			<td>Corning</td>
			<td>357070</td>
			<td>*Falcon brand</td>
		</tr>
		<tr>
			<td>Eppendorf tubes (1.5 ml)</td>
			<td>Corning</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml polystyrene round bottomed tubes</td>
			<td>Corning</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Tools</td>
			<td>Fine Science Tools&#160;</td>
			<td>*Various&#160;</td>
			<td>*Dissection scissors, dumont forcep and standard forcep&#160;</td>
		</tr>
		<tr>
			<td>Hemocytometer&#160;</td>
			<td>Richert</td>
			<td>1490</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 100 x 15 mm Petri Dish</td>
			<td>Corning</td>
			<td>351029</td>
			<td>*Falcon brand</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Thermo Fisher</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Chloride</td>
			<td>BDH</td>
			<td>BDH0208-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher Bioreagents</td>
			<td>BP1600-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>Sigma</td>
			<td>B7651-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E5134-1KG</td>
			<td>Ethylenediaminetetraacetic acid</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution</td>
			<td>Thermo Fisher</td>
			<td>14175-095&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher</td>
			<td>15630-080</td>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G8540-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS Ultrapure</td>
			<td>Invivogen</td>
			<td>tlrl-3pelps</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution&#160;</td>
			<td>Thermo Fisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 100x</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>BDH</td>
			<td>BDH0268-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>BDH</td>
			<td>BDH9258-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant GM-CSF</td>
			<td>Peprotech</td>
			<td>315-03-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Rooster Comb Sodium Hyaluronate&#160;</td>
			<td>Sigma</td>
			<td>H5388-1G</td>
			<td>*Used to make fluoresceinated hyaluronan</td>
		</tr>
		<tr>
			<td>RPMI-1640&#160;</td>
			<td>Thermo Fisher</td>
			<td>21870-076</td>
			<td>No sodium pyruvate no glutamine. Warm media to 37oC before using.&#160;</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher&#160;</td>
			<td>5271-10&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Sigma</td>
			<td>50876-1Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma</td>
			<td>P5290-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Fisher Bioreagents</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Fc Receptor (unlabeled), Tissue Culture Supernatant</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Clone: 2.4G2</td>
		</tr>
		<tr>
			<td>Anti-CD11c PeCy7</td>
			<td>eBioscience</td>
			<td>25-0114-82</td>
			<td>Clone: N418</td>
		</tr>
		<tr>
			<td>Anti-Gr-1 efluor450</td>
			<td>eBioscience</td>
			<td>48-5931-82</td>
			<td>Clone: RB6-8C5</td>
		</tr>
		<tr>
			<td>Anti-MHCII APC</td>
			<td>eBioscience</td>
			<td>17-5321-82</td>
			<td>Clone: M5/114.15.2</td>
		</tr>
		<tr>
			<td>Biotinylated Anti-MerTK</td>
			<td>Abcam</td>
			<td>BAF591</td>
			<td>Goat polyclonal IgG</td>
		</tr>
		<tr>
			<td>Streptavidin PE</td>
			<td>eBioscience</td>
			<td>12-4317-87</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma</td>
			<td>P4170-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole)</td>
			<td>Sigma</td>
			<td>D9542-5MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and identification of gm-csf derived alveolar-like macrophages and dendritic cells from mouse bone marrow

मैक्रोफेज और वृक्ष के समान कोशिकाओं (डीसी) सहज प्रतिरक्षा ऊतकों और अंगों लसीकावत् कि रोगजनकों के खिलाफ रक्षा करने में एक महत्वपूर्ण भूमिका निभा में पाया कोशिकाओं रहे हैं। हालांकि, वे पर्याप्त संख्या में अलग-थलग करने के लिए उन्हें विस्तार से अध्ययन करने के लिए मुश्किल हो जाता है, इसलिए, इन विट्रो मॉडल विकसित किया गया है। अस्थि मज्जा व्युत्पन्न मैक्रोफेज और वृक्ष के समान कोशिकाओं की इन विट्रो संस्कृतियों प्रतिरक्षा अध्ययन के लिए अच्छी तरह से स्थापित और मूल्यवान तरीके हैं। इधर, संवर्धन और साइटोकाइन granulocyte बृहतभक्षककोशिका कॉलोनी उत्तेजक कारक (जीएम-सीएसएफ) का उपयोग प्राथमिक माउस अस्थि मज्जा की कोशिकाओं का एक ही संस्कृति से दोनों डीसी और मैक्रोफेज की पहचान करने के लिए एक विधि का वर्णन किया है। इस प्रोटोकॉल की स्थापना की प्रक्रिया पहले लुट्ज़ एट अल द्वारा विकसित पर आधारित है। अस्थि मज्जा व्युत्पन्न DCs के लिए 1999 में। संस्कृति विषम है, और MHCII और fluoresceinated हयालूरोनान (FL-हा) अपरिपक्व और परिपक्व डीसी से मैक्रोफेज भेद करने के लिए इस्तेमाल कर रहे हैं। ये जीएम- CSF व्युत्पन्न मैक्रोफेज समर्थकइन विट्रो व्युत्पन्न मैक्रोफेज है कि निकट दोनों phenotype और समारोह में वायुकोशीय मैक्रोफेज के समान के लिए एक सुविधाजनक स्रोत ख़बरदार।

इस विधि के प्रमुख कदम का सारांश एक फ़्लोचार्ट चित्र 1 में दिखाया गया है। संस्कृति के अलग अलग समय पर घनत्व और अस्थि मज्जा संस्कृति की आकृति विज्ञान चित्रा 2 में दिखाया जाता है। 1 ?...

Watch this Scientific Journal Video about Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow at JoVE.com

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

जनरेशन और जीएम-सीएसएफ की पहचान व्युत्पन्न वायुकोशीय तरह मैक्रोफेज और वृक्ष के समान माउस अस्थि मज्जा से कोशिकाओं

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

The overall goal is this procedure is to generate and distinguish alveolar-like macrophages from dendritic cells in in vitro murine bone marrow cell cultures supplemented with GM-CSF. This in vitro method can help answer key questions regarding the function of dendritic cells or macrophages. The main advantage of the technique is that it can generate sufficient cell numbers and it can distinguish between dendritic cells and alveolar-like macrophages.The implications of this technique may extend towards the therapy of pulmonary proteinosis as it facilitates the acquisition of alveolar-like macrophages, which can be used to treat this disease. Generally, individuals new to this method will need practice with isolating the cleaned bones and flushing the bone marrow. Visual demonstration of this method is useful as not all techniques used as easily understood by written instruction alone.15 minutes before beginning the dissection, turn on a biological safety cabinet to purge the cabinet air, and to stabilize the air flow, and clean the cabinet surface with 70%ethanol. When the cabinet is ready, place a mouse on top of one to two paper towels in the prone position and spray the animal with 70%ethanol. Then, with the non-dominant hand, lift the skin around the thoracic area and use scissors to make an incision.Grasping both ends of the incision, carefully pull until the two ends of the incision meet at the stomach. Then place the mouse in the supine position and use one hand to pull the lower half of the skin down until the legs are exposed. Holding the foot, cut the Achilles tendons above the ankle joint and the ligaments connecting the muscles to the foot at the lower end of the tibia to loosen the foot from the leg bones.Keeping the leg raised, remove the muscles and ligaments around the knee joint at the lower end of the femur to severe the thigh muscles from the lower leg. Using forceps, gently pull the loosened muscles away from the fibular and femoral surfaces. Then pressing the scissors in the open position at the hip joint, rotate the leg and gently pull on the femur to separate the leg bone from the hip joint while cutting the leg free from the body.Now holding the leg at the hip end with a sterile forceps, use another forceps to remove the foot from the ankle end. Then holding the distal end of the femur with one forceps and the proximal end of the tibia and fibula with another, carefully bend the tibia and fibula in the opposite direction of the knee joint to separate the lower leg from the femur and patella. Use the forceps to remove the remaining ligaments, muscles, and fibula from the lower leg bones and place the cleaned tibia on an inverted Petri dish lid, containing one to two milliliters of HBSS/FCS.Holding the lower end of the femur with one set of forceps and the patella with another, bend the knee and surrounding tissues in the opposite direction to remove the joint. Then remove any of the remaining connective tissues from the femur and place the bone on the inverted Petri dish lid. When all of the bones have been harvested, use forceps to clean any residual tissue still attached to the bones and immerse the bones in 10 milliliters of HBSS/FCS within the base of the Petri dish.Keeping the dish partially shielded with a sterile lid, cut each of the cleaned bones in half with scissors. Then use a one-milliliter syringe, equipped with a 26.5-gauge needle to flush one milliliter of HBSS/FCS from the dish into the end of each bone piece until all of the red marrow has been liberated. Pass any visible clumps of bone marrow through the syringe several times to form a single cell suspension, followed by thorough mixing with a 10 milliliter pipette.Transfer the cells to a collection tube and rinse the Petri dish one time with five milliliters of HBSS/FCS to collect any residual cells. Then pool the wash in the collection tube and place the cells on ice. To set up a bone marrow cell culture, centrifuge the collected cells and aspirate the supernatant with a sterile glass Pasteur pipette attached to vacuum suction.Loosen the red blood cell-rich pellet with tapping. Then add 10 milliliters of RBC lysis buffer and resuspend the cells with vortexing. After a five-minute, room temperature incubation, arrest the lysis with 10 milliliters of HBSS/FCS and spin down the cells, resuspending the pellet in bone marrow cell medium.Count the number of viable cells by Trypan Blue exclusion and transfer the needed cells into a new tube with bone marrow cell medium. Then collect the cells by centrifugation and resuspend the pellet at a four million cell per milliliter dilution in bone marrow cell medium with 20 nanograms per milliliter of GM-CSF. Next, add 9.5 milliliters of bone marrow cell medium supplemented with 20 nanograms per milliliter of GM-CSF to one sterile bacterial Petri dish per culture and add 500 microliters of cells to the center of each dish.Incubate the cells at 37 degrees Celsius and five percent CO2. On day three, add 10 milliliters of fresh bone marrow medium supplemented with 20 nanograms per milliliter of GM-CSF to each culture, taking care to minimize any disturbances to the cells. On day six, carefully transfer 10 milliliters of cell culture supernatant to a sterile 50 milliliter conical tube.Centrifuge the cells and resuspend the pellet in 10 milliliters of fresh bone marrow cell medium, containing 20 nanograms per milliliter of GM-CSF. Then gently vortex the cell suspension and return the cells to the culture dish. On day one, the bone marrow cells are small and sparse but by day three, the cells have increased in number and size, and some have begun to adhere.By day six, there are more cells and both adherent and non-adherent fractions are observed. The culture can be harvested from day seven to 10 and gated by size and live cells with a higher percentage of CD11c-positive cells obtained from day 10 cultures. At day seven, the collected non-adherent fraction is greatly enriched for cells with a dendritic morphology.The CD11c-positive, GR1-negative populations, harvested on day seven and 10, can be divided into three main populations. Macrophages, immature dendritic cells, and mature dendritic cells by MHCII expression and FL-HA binding. Interestingly, MerTK, although considered to be a macrophage marker, exhibits a robust expression on both macrophage and immature dendritic cell populations, harvested on days seven and 10, with a low level of expression observed on mature dendritic cells.While attempting this procedure, it is important to remember to use sterile techniques throughout the bone marrow harvest and cell culture. Following this procedure, the cells also can be sorted to examine cell populations separately, or they can be stimulated with inflammatory agents and subsequently analyzed for their cytokine production by intracellular staining and flow cytometry. After watching this video, you should have a good understanding of how to isolate bone marrow cells and culture them in GM-CSF to generate both dendritic cells and alveolar-like macrophages.

Research

JoVE Journal

Immunology and Infection

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

सक्रिय अस्थि मज्जा व्युत्पन्न मैक्रोफेज और Bradyzoite स्थितियां में Toxoplasma gondii पुटी दीवार संरचना

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

एक फ्री सेल परख जीएम-CSF एंटीबॉडी के उदासीन क्षमता का आकलन प्रणाली पुनः संयोजक घुलनशील रिसेप्टर जीएम-CSF का उपयोग

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

जनरेशन और ट्रैकिंग अध्ययन के लिए Qdot Nanocrystals साथ murine अस्थि मज्जा व्युत्पन्न वृक्ष के समान कोशिकाओं के लेबल

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

अलगाव और और माउस आंत से वृक्ष के समान कोशिकाओं मैक्रोफेज की विशेषता

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

अस्थि मज्जा व्युत्पन्न मैक्रोफेज का उपयोग मैक्रोफेज ध्रुवीकरण की जांच

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

Bone Marrow-derived Macrophage Production

अस्थि मैक्रोफेज उत्पादन मज्जा व्युत्पन्न

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

माउस अस्थि मज्जा से Plasmacytoid वृक्ष के समान कोशिकाओं की शुद्धि के लिए प्रतिदीप्ति सक्रिय सेल छंटनी

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

पूरे रक्त से मानव monocyte व्युत्पन्न वृक्ष के समान कोशिकाओं की पीढ़ी

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

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

पहचान और Oligopotent और वंश की अलगाव-प्रतिबद्ध माइलॉयड Progenitors से माउस अस्थि मज्जा

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

चूहों से अस्थि मज्जा-व्युत्पन्न डेंड्राइटिक कोशिकाओं को अलग करने और खेती करने के लिए किफायती और कुशल प्रोटोकॉल

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...