Name: identification and isolation of oligopotent and lineage-committed myeloid progenitors from mouse bone marrow
Uploaded: 2018-07-29T15:00:00
Description: Watch this Scientific Journal Video about Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow at JoVE.com

Denne protokol blev udviklet ved hjælp af midler fra Board af guvernører regenerativ medicin Institut på Cedars-Sinai Medical Center (til HSG), en karriere i immunologi fellowship fra American Association for Immunologists (til AY og HSG) og en Scholar Award fra den American Society of Hematology (til AY). Vi takker Flow flowcytometri Core på Cedars-Sinai Medical Center for bistand med FACS sortering.

Alle metoder, der beskrives her blev godkendt af institutionelle Animal Care og brug udvalg (IACUC) af Cedars-Sinai Medical Center.
1. isolering af musen knoglemarven og forberedelse af en enkelt cellesuspension
<ol><li>Aflive mus efter institutionelle retningslinjer.</li>
	<li>Placer de aflivede mus på ryggen og spray det med 70% ethanol (EtOH). Lave en lille (3-5 mm) snit i hver hind lemmer på ankel niveau bruger skarpe, spidse sakse, og træk huden op mod kroppen til at fjerne det fra b...

<ol>
	<li>Akashi, K., Traver, D., Miyamoto, T., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clonogenic+common+myeloid+progenitor+that+gives+rise+to+all+myeloid+lineages.">A clonogenic common myeloid progenitor that gives rise to all myeloid lineages.</a> Nature. 404 (6774), 193-197 (2000).</li><li>Y&#225;&#241;ez, A., Ng, M. Y., Hassanzadeh-Kiabi, N., Goodridge, 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=IRF8+acts+in+lineage-committed+rather+than+oligopotent+progenitors+to+control+neutrophil+vs+monocyte+production.">IRF8 acts in lineage-committed rather than oligopotent progenitors to control neutrophil vs monocyte production.</a> Blood. 125 (9), 1452-1459 (2015).</li><li>Auffray, 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=CX3CR1++CD115++CD135++common+macrophage/DC+precursors+and+the+role+of+CX3CR1+in+their+response+to+inflammation.">CX3CR1+ CD115+ CD135+ common macrophage/DC precursors and the role of CX3CR1 in their response to inflammation.</a> Journal of Experimental Medicine. 206 (3), 595-606 (2009).</li><li>Fogg, D. 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=A+clonogenic+bone+marrow+progenitor+specific+for+macrophages+and+dendritic+cells.">A clonogenic bone marrow progenitor specific for macrophages and dendritic cells.</a> Science. 311 (5757), 83-87 (2006).</li><li>Onai, N., 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=Identification+of+clonogenic+common+Flt3+M-CSFR++plasmacytoid+and+conventional+dendritic+cell+progenitors+in+mouse+bone+marrow.">Identification of clonogenic common Flt3+M-CSFR+ plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow.</a> Nature Immunology. 8 (11), 1207-1216 (2007).</li><li>Hettinger, 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=Origin+of+monocytes+and+macrophages+in+a+committed+progenitor.">Origin of monocytes and macrophages in a committed progenitor.</a> Nature Immunology. 14 (8), 821-830 (2013).</li><li>Y&#225;&#241;ez, 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=Granulocyte-monocyte+progenitors+and+monocyte-dendritic+cell+progenitors+independently+produce+functionally+distinct+monocytes.">Granulocyte-monocyte progenitors and monocyte-dendritic cell progenitors independently produce functionally distinct monocytes.</a> Immunity. 47 (5), 890-902 (2017).</li><li>Olsson, 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=Single-cell+analysis+of+mixed-lineage+states+leading+to+a+binary+cell+fate+choice.">Single-cell analysis of mixed-lineage states leading to a binary cell fate choice.</a> Nature. 537 (7622), 698-702 (2016).</li><li>Amend, S. R., Valkenburg, K. C., Pienta, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+hind+limb+long+bone+dissection+and+bone+marrow+isolation.">Murine hind limb long bone dissection and bone marrow isolation.</a> Journal of Visualized Experiments. (110), (2016).</li><li>Y&#225;&#241;ez, A., Goodridge, 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=Interferon+regulatory+factor+8+and+the+regulation+of+neutrophil,+monocyte,+and+dendritic+cell+production.">Interferon regulatory factor 8 and the regulation of neutrophil, monocyte, and dendritic cell production.</a> Current Opinion in Hematology. 23 (1), 11-17 (2016).</li><li>Manz, M. G., Miyamoto, T., Akashi, K., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+isolation+of+human+clonogenic+common+myeloid+progenitors.">Prospective isolation of human clonogenic common myeloid progenitors.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (18), 11872-11877 (2002).</li></ol>

Weissman gating strategi for musen myeloide stamceller identifikation1 har været guldstandarden for immunologists og Hematologer for næsten 20 år, men det er nu tydeligt, at "CMP" og "GMP" gates er meget heterogen og mere præcise gating strategier er nødvendige. Den protokol, som vi har beskrevet her tillader identifikation af oligopotent og afstamning-begået delmængder i C57BL/6J mus for mere præcis kvantificering af specifikke myeloide stamceller og kortlægning af myelopoiesis veje, sam...

Neutrofile og monocytter og dendritiske celler (DCs) er myeloide celler, der opstår fra hæmatopoietisk progenitorceller, primært i knoglemarven, ved en proces kaldet myelopoiesis. Fælles myeloide stamceller (Kystforvaltningsplaner) har potentiale til at producere myeloide celler, samt megakaryocytes og erytrocytter, men ikke lymfoide celler. Granulocyt-monocyt stamfaderen (GMPs), som er afledt af Kystforvaltningsplaner, producere granulocytter og monocytter, men har mistet megakaryocyte og erytrocyt potentielle. Monocytter og klassisk og plasmacytoid DCs (cDCs/fremdaterede) menes også at opstå fra fælles ophav kendt som monocyt-DC stamfaderen (MDP'er), som er produceret af Kystforvaltningsplaner. gradvis begrænsning af lineage potentiale i sidste ende resulterer i slægt-begået ophav: granulocyt progenitorceller, monocyt ophav og dendritiske celle stamfaderen (figur 1).
Weissman og kolleger rapporteret at Kystforvaltningsplaner findes i Lin- c-Kit+ Sca-1- (LKS-) CD34+ FcγRlo brøkdel af musen knoglemarven, mens GMPs er indeholdt i LKS- CD34+ FcγR Hej fraktion1. Men disse "CMP" og "GMP" brøker er meget heterogen. For eksempel, indeholder "GMP" brøkdel også afstamning-begået granulocyt ophav og monocyt stamfaderen1,2. MDP'er separat blev rapporteret til at være CX3CR1+ Flt3+ CD115+ progenitorceller, som også udtrykker CD34 og FcγR3,4. MDP'er give anledning til cDC/pDC-producerende fælles DC stamfaderen (CDPs), som er blevet rapporteret at udtrykke lavere niveauer af c-Kit (CD117) og er ikke inkluderet i LKS- brøkdel5.
Tidligere var det antaget at monocytter opstår via en enkelt vej (CMP-GMP-MDP-monocyt). Overensstemmelse med denne model, monocyt-begået stamfaderen produceret af GMPs (opkaldt monocyt progenitorceller, MPs)2 og MDP'er (opkaldt fælles monocyt progenitorceller, cMoPs)6 synes at være de samme celler på grundlag af fælles overflade markør udtryk . Dog, vi for nylig demonstreret at monocytter produceres uafhængigt af GMPs og MDP'er, og var i stand til at skelne mellem parlamentsmedlemmer og cMoPs af encellede RNA sekventering7.
Vi har for nylig ændret Weissman "CMP" og "GMP" gating strategi for at identificere 6 subfractions af C57BL/6J mus knoglemarv indeholdende forskellige oligopotent og afstamning-begået myeloide stamceller undersæt. Vi første gang rapporteret, at farvning for Ly6C og CD115 tillader dyrkning af oligopotent GMPs, samt granulocyt stamfaderen (GPs) og monocyt stamfaderen (både MPs og cMoPs, som vi er i øjeblikket ude af stand til at adskille) fra "GMP" fraktion2 (LKS - CD34+ FcγRHej gate; Figur 1). Vi viste efterfølgende, at MDP'er findes overvejende i "CMP" fraktion (LKS- CD34+ FcγRlo gate), som også indeholder Flt3+ CD115lo og Flt3- delmængder7 (figur 1 ). CMP-Flt3+ CD115lo brøkdel udbytter både GMPs og MDP'er ved adoptiv overførsel. CMP-Flt3- delmængde indeholder stamfaderen, der synes at være mellemprodukter mellem CMP-Flt3+ CD115lo celler og GMPs. I modsætning til MDP'er besidder både den CMP-Flt3+ CD115lo og CMP-Flt3- fraktioner også megakaryocyte og erytrocyt potentielle.
Det er vigtigt at bemærke, at det er i øjeblikket uklart, om "CMP" fraktioner indeholder stamfaderen, der virkelig er oligopotent (f.eks. individuelle celler i CMP-Flt3+ CD115lo brøkdel der besidder neutrofile, monocyt, DC, megakaryocyte og erytrocyt potentielle), eller alternativt, omfatter en blanding af stamfaderen med mere begrænset lineage potentiale. Kolonidannende assays (methylcellulose kulturer) afslørede celler med granulocyt (neutrofile), erytrocyt, monocyt og megakaryocyte potentielle (CLAUSS celler) i "CMP", CMP-Flt3+ CD115lo og CMP-Flt3- fraktioner1 ,7, men tillader ikke vurderingen af DC potentiale. Derimod kolonidannende assays påvist eksistensen af oligopotent GMPs (ophav med både neutrofile og monocyt potentielle) i "GMP" fraktion1,2, og dette er understøttet af de seneste single-celle transkriptom analyse8. Det vides i øjeblikket ikke, men om disse oligopotent GMPs også producere andre granulocytter (eosinofile, basofile og mastceller).
Baseret på disse undersøgelser, vi nu demonstrere kan hvordan 7 overflade markører (c-Kit, Sca-1, CD34, FcγR, Flt3, Ly6C og CD115) bruges til at identificere og isolere disse 6 delmængder af oligopotent og afstamning-begået myeloide stamceller. Protokollen beskrevet her kan anvendes til in vitro- kultur assays (methylcellulose eller flydende kulturer), i vivo adoptiv overførsel eksperimenter i mus og molekylær analyse (bulk og encellede RNA sekventering, Western blotting, etc.).
Protokollen består af 3 faser: 1) udarbejdelse af en enkelt cellesuspension af knoglemarv celler, 2) berigelse for hæmatopoietisk stamfaderen (magnetisk aktiveret celle sortering), og 3) identifikation og isolation, hvis det ønskes af stamfader delmængder af flow flowcytometri (ved hjælp af et analyseapparat eller et sorteringsanlæg, som passende). Det første skridt er isolering af knoglemarv celler fra lårben og skinneben af aflivede mus og svarer til andre tidligere beskrevet protokoller9. Næste, prøven er beriget til stilken og stamfader celler ved hjælp af en cocktail af antistoffer mod celle overflade markører af erytrocytter, neutrofiler, monocytter, lymfocytter, osv., nedbryder de differentierede celler. Dette er ikke obligatorisk, men anbefales kraftigt at optimere påvisning af stamfader delmængder og reducere mængden af antistoffer til stamfader identifikation og den nødvendige tid til flowcytometri. Lineage udtynding protokol nedenfor beskriver Magnetic-Activated celle sortering (MLA) ved hjælp af en mus afstamning celle nedbrydningen Kit (som indeholder biotinylated antistoffer mod CD5, CD45R (B220), CD11b, Gr-1 (Ly6G/C), 7-4, og Ter-119, plus anti-biotin microbeads) og en automatiseret magnetisk separator. Det sidste trin er at identificere (og sortering, hvis det ønskes) af stamfader delmængder ved flowcytometri. Panelet antistof beskrevet nedenfor (Se også tabel 1) er udviklet til at blive brugt i et flow forskellige (analyzer eller sorteringsanlæg) med 4 lasere (405 nm, 488 nm, 561 nm, 640 nm).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58061/58061fig1.jpg"> Figur 1: neutrofile, monocyt og DC ophav og differentiering veje. Den seneste reviderede model af myelopoiesis7 illustreres med Weissman gates for "Kystforvaltningsplaner" (blå) og "GMPs" (grøn)1 overlejret. Dette tal er blevet ændret fra Yáñez et al. 20177. <a href="https://www.jove.com/files/ftp_upload/58061/58061fig1large.jpg" target="_blank">Venligst klik her for at se en større version af dette tal.</a>

<table border="0" cellpadding="0" cellspacing="0" width="945">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Mouse: Wild-type C57BL/6J (CD45.2)</td>
			<td style="width:177px;">The Jackson Laboratories</td>
			<td style="width:172px;">Cat#JAX:000664</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Lineage Cell Depletion Kit, mouse</td>
			<td style="width:177px;">Miltenyi Biotec</td>
			<td style="width:172px;">Cat#130-090-858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Rat anti-mouse CD34 (clone RAM34) FITC</td>
			<td style="width:177px;">BD Biosciences</td>
			<td style="width:172px;">Cat#553733</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">Rat anti-mouse CD16/CD32 (Fc&#947;R; clone 93) APC-Cy7</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#101327</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">Rat anti-mouse Ly6A/E (Sca-1; clone 108113) PE-Cy7</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#108114</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:357px;">Rat anti-mouse CD117 (c-Kit; clone 2B8) Pacific Blue</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#105820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Rat anti-mouse Ly6C (clone HK1.4) PerCP-Cy5.5</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#128012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Rat anti-mouse CD115 (clone AFS98) PE</td>
			<td style="width:177px;">BioLegend</td>
			<td style="width:172px;">Cat#135506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">Rat anti-mouse CD135 (Flt3; clone A2F10.1) APC</td>
			<td style="width:177px;">BD Biosciences</td>
			<td style="width:172px;">Cat#560718</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">CountBright Absolute Counting Beads</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:172px;">Cat#C36950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">AutoMACS Separator</td>
			<td style="width:177px;">Miltenyi Biotec</td>
			<td style="width:172px;">N/A</td>
			<td>Use the &#34;deplete&#34; program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">BD LSRFortessa</td>
			<td style="width:177px;">BD Biosciences</td>
			<td style="width:172px;">N/A</td>
			<td>5 lasers, 15 colors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">BD FACS Aria III cell sorter</td>
			<td style="width:177px;">BD Biosciences</td>
			<td style="width:172px;">N/A</td>
			<td>5 lasers, 13 colors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:357px;">FlowJo</td>
			<td style="width:177px;">FlowJo, LLC</td>
			<td style="width:172px;">https://www.flowjo.com</td>
			<td>For further analysis of the .fcs files</td>
		</tr>
	</tbody>
</table>

identification and isolation of oligopotent and lineage-committed myeloid progenitors from mouse bone marrow

Myeloide stamceller, der giver neutrofiler, monocytter og dendritiske celler (DCs) kan identificeres i og isoleret fra knoglemarven af mus til hematological og immunologiske analyser. For eksempel, kan undersøgelser af de cellulære og molekylære egenskaber af myeloide stamceller befolkninger afsløre underliggende leukemic transformation mekanismer, eller demonstrere hvordan immunsystemet reagerer på patogen eksponering. Tidligere beskrevet flow flowcytometri strategier for myeloide stamceller identifikation har gjort betydelige fremskridt på mange områder, men fraktioner de identificere er meget heterogen. De mest almindeligt anvendte gating strategier definere knoglemarv fraktioner, der er beriget til de ønskede befolkninger, men også indeholder store mængder af "forurener" stamfaderen. Vores nylige undersøgelser har løst en stor del af denne forskelligartethed, og den protokol, vi præsenterer her tillader dyrkning af 6 delpopulationer af oligopotent og afstamning-begået myeloide stamceller fra 2 tidligere beskrevet knoglemarv fraktioner. Protokollen beskriver 3 stadier: 1) isolering af knoglemarv celler, 2) berigelse for hæmatopoietisk stamfaderen ved magnetisk aktiveret celle sortering (lineage udtynding af MLA), og 3) identifikation af myeloide stamceller delmængder af flowcytometri (herunder Fluorescens-aktiveret celle sortering, FACS, hvis det ønskes). Denne fremgangsmåde tillader stamfader kvantificering og isolation for en række in vitro og i vivo applikationer, og har allerede givet roman indsigt i veje og mekanismer af neutrofile, monocyt og DC differentiering.

Ved hjælp af protokollen, der er beskrevet ovenfor, er det muligt at opnå ~ 100 millioner celler (herunder røde blodlegemer, eller ~ 50 millioner nukleeret celler) fra både lårben og skinneben (2 ben) af en C57BL/6J mus (6-8 uger gammel, mand eller kvinde). 1-2 millioner Lin- celler kan isoleres pr. mus af MACS udtynding af Lin+ celler.
Hver af de 6 myeloide stamceller delmængder udgør ~ 1-4% af Lin<...

Watch this Scientific Journal Video about Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow at JoVE.com

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

Vi demonstrere, hvordan til at identificere og isolere 6 delmængder af myeloide stamceller fra murine knoglemarven ved hjælp af en kombination af magnetiske og fluorescens sortering (MACS og FACS). Denne protokol kan bruges til i vitro kultur assays (methylcellulose eller flydende kulturer), i vivo adoptiv overførsel eksperimenter og RNA/protein analyser.

Identifikation og isolering af Oligopotent og afstamning-begået myeloide ophav fra mus knoglemarv

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

This method could be helpful for hematologists and immunologists studying the production and function of myeloid cells, including neutrophils, monocytes, and dendritic cells. The main advantage of this technique is that it enables more precise identification of myeloid progenitor subsets than previous strategies. To enrich bone marrow cells for progenitors by magnetic-activated cell sorting, or MACS, pellet the cells by centrifugation and resuspend the pellet in 40 microliters of MACS staining buffer per one times 10 to the seven bone marrow cells.To deplete differentiated lineage positive cells, add 10 microliters of biotin antibody cocktail per one times 10 to the seven cells and to mix by pipetting for a 10 minute incubation at four degrees Celsius. At the end of the incubation, add 30 microliters of staining buffer per one times 10 to the seven cells with mixing, followed by 20 microliters of anti-biotin microbeads per one times 10 to the seven cells. After 15 minutes at four degrees Celsius, wash the cells with one milliliter of staining buffer per one times 10 to the seven cells and resuspend the pellet in 500 microliters of fresh staining buffer for up to one times 10 to the eight cells.Next, set up the automated magnetic separator according to the manufacturer's instructions and place the tube of labeled cells into the separator. Begin the negative selection program to collect the negative fraction which contains the progenitor enriched lineage negative cells. Pellet the lineage negative cells by centrifugation and resuspend the pellet, which is now white, in two milliliters of staining buffer per mouse for counting.To isolate the myeloid progenitor cells by FACS, add one times 10 to the fifth lineage negative cells to each of eight control microcentrifuge tubes for voltage selection and color compensation and add the rest of the cell sample to a ninth microcentrifuge tube for staining with all seven of the surface marker antibodies of interest for progenitor identification and sorting. Pellet the cells by centrifugation and resuspend the control pellets in 100 microliters of FACS staining buffer and the experimental sample pellet in 100 microliters of staining buffer per five times 10 to the six cells. Next, add anti-CD16/CD32 antibody to the Fc gamma receptor single stain tube and the sample tube.Vortex gently and incubate the samples for 10 minutes at four degrees Celsius. At the end of the incubation, add the other antibodies to the appropriate corresponding single stain tubes and to the sample tube and after gentle vortexing, incubate the samples for 15 minutes at four degrees Celsius. At the end of the incubation, add 900 microliters of staining buffer to the control tubes and one milliliter of staining buffer per one times 10 to the seven cells to the sample tube for centrifugation.Resuspend the control pellets in 200 microliters of fresh staining buffer per tube and the experimental pellet in 500 microliters per 2.5 times 10 to the seven cells and transfer the labeled cells to individual corresponding five milliliter FACS tubes. Then set up the flow cytometer according to standard protocols and use the unstained and single stained controls to set the voltages and the color compensations. To identify and isolate the myeloid progenitor cells, load the experimental cell sample onto the cytometer and create a forward scatter area versus side scatter area plot, gating to exclude the debris and dead cells.Create a forward scatter height versus forward scatter width plot of the live cells and gate to exclude the doublets. Repeat this process with a side scatter height versus side scatter width plot of the forward scatter singlets and gate to exclude the doublets. Create a Sca-1 versus c-Kit plot and gate to select the c-Kit positive Sca-1 negative progenitors.Then create a CD-34 versus FC gamma receptor plot and gate to select the mixed common myeloid progenitor and mixed granulocyte monocyte progenitor populations. It's important to gate the mixed common myeloid progenitor and mixed granulocyte monocyte progenitor populations as precisely as possible. It can be helpful to use a pseudo-color density plot or a contour plot for more accurate gating.Use the cells in the mixed common myeloid progenitor gate to create a CD115 versus Flt3 plot and gate to select the common myeloid progenitor Flt3 positive CD115 low cells, the common myeloid progenitor Flt3 negative CD115 low cells, and the Flt3 positive CD115 high monocyte dendritic cell progenitors. Use the cells in the mixed granulocyte monocyte progenitor gate to create a Ly6C versus FC Gamma receptor plot and gate to select the Ly6C negative cells and the Ly6C positive cells. Then create a CD115 versus Flt3 plot for each gated mixed granulocyte monocyte progenitor cell subpopulation and gate the Ly6C negative Flt3 negative CD115 low granulocyte monocyte progenitors, the Ly6C positive Flt3 negative CD115 low granulocyte progenitors, and the Ly6C positive Flt3 negative CD115 high monocyte progenitors.Max lineage depletion is efficient at depleting differentiated cells and for enriching for the c-Kit positive progenitor cells. The lineage negative fraction does still contain some c-Kit negative cells, but these cells will be eliminated in subsequent flow cytometry gating steps. Progenitor yields after FACS sorting can vary depending on the sorter settings used, but it should be possible to obtain between one to four times 10 to the fourth cells per fraction from each mouse with a post-sort purity of greater than 95%for each fraction.Good staining is critical for clean separation of the populations. You may need to titrate the antibodies or choose different fluorophores to ensure optimal separation. This protocol can be used to identify progenitor cells to assess differences in bone marrow composition between samples, to isolate progenitors for molecular analysis or to assess their ability to produce myeloid cells.

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Immunology and Infection

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Isolation, rensning og mærkning af museknoglemarv Neutrophils for Funktionelle Studier og adoptiv overføring Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Isolering af myeloide dendritiske celler og epitelceller fra human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Bone Marrow-derived Macrophage Production

Bone Marrow-afledt Macrophage Production

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

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

Isolation og intravenøs injektion af murine knoglemarv Afledte Monocytter

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Tracking Mouse Bone Marrow Monocytes In Vivo

Sporing Mouse knoglemarv Monocytter<em&gt; In Vivo</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Myeloid Cell Isolation fra Mouse Hud og drænende lymfeknude Efter Intrakutan Vaccination med levende svækket<em&gt; Plasmodium</em&gt; sporozoitter

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Murine Hind Limb Lang Bone Dissektion og knoglemarv Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

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

Generation og Identifikation af GM-CSF Afledt Alveolære-lignende makrofager og dendritiske celler fra mus Bone Marrow

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. ...

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

Fluorescens-aktiveret cellesortering til oprensning af Plasmacytoide dendritiske celler fra knoglemarven i mus

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

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Generation af stort antal myeloide stamceller og dendritiske celle prækursorer fra Murine knoglemarven ved hjælp af en roman celle sortering strategi

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...