Name: identification of erythromyeloid progenitors and their progeny in the mouse embryo by flow cytometry
Uploaded: 2017-07-17T12:30:00
Description: Watch this Scientific Journal Video about Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry at JoVE.com

Forfatterne takker Prof Frederic Geissmann og Prof Christian Schulz for indgående drøftelser; Dr Hannah Garner for kritisk læsning af manuskriptet, dr. Xavier Montagutelli og dr. Jean Jaubert og personalet på Institut Pasteur dyreanlæg til støtte med musemestring; Og Pascal Dardenne og Vytaute Boreikaite, en Amgen Scholar, til deres tekniske bistand. Forskning i EGP-laboratoriet finansieres af Institut Pasteur, CNRS, Cercle FSER (FRM og en startpakke fra Institut Pasteur og REVIVE-konsortiet. LI støttes af et ph.d.-stipendium fra REVIVE-konsortiet.

Dyreprocedurer blev udført i overensstemmelse med det godkendte institut for dyrepleje og brug af Institut Pasteur (CETEA). 1. I Utero Pulse Mærkning i Csf1r MeriCreMer Rosa LSL-YFP Embryoer <ol><li> Forbered stamopløsningen af ​​4-hydroxytamoxifen (OH-TAM, 50 mg / ml). <ol><li> Åbn hætteglasset med 25 mg OH-TAM under fumehood og tilsæt 250 μL ethanol (100%). </li><li> Overfør OH-TAM-opløsninge...

<ol>
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E., Palis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitive+Hematopoiesis+in+the+Yolk+Sac+Emerges+from+Wnt-Responsive+Hemogenic+Endothelium+Independently+of+Circulation+and+Arterial+Identity.">Definitive Hematopoiesis in the Yolk Sac Emerges from Wnt-Responsive Hemogenic Endothelium Independently of Circulation and Arterial Identity.</a> Stem Cells. , (2015).</li><li>Kieusseian, A., Brunetdela de la Grange, P., Burlen-Defranoux, O., Godin, I., Cumano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immature+hematopoietic+stem+cells+undergo+maturation+in+the+fetal+liver.">Immature hematopoietic stem cells undergo maturation in the fetal liver.</a> Development. 139 (19), 3521-3530 (2012).</li><li>Gomez Perdiguero, ., E, , 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=Tissue-resident+macrophages+originate+from+yolk-sac-derived+erythro-myeloid+progenitors.">Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.</a> Nature. 518 (7540), 547-551 (2015).</li><li>Hoeffel, G., 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=C-Myb(+)+erythro-myeloid+progenitor-derived+fetal+monocytes+give+rise+to+adult+tissue-resident+macrophages.">C-Myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages.</a> Immunity. 42 (4), 665-678 (2015).</li><li>Kierdorf, K., Prinz, M., Geissmann, F., Gomez Perdiguero, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+function+of+tissue+resident+macrophages+in+mice.">Development and function of tissue resident macrophages in mice.</a> Semin Immunol. 27 (6), 369-378 (2015).</li><li>Houssaint, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+the+mouse+hepatic+primordium.+II.+Extrinsic+origin+of+the+haemopoietic+cell+line.">Differentiation of the mouse hepatic primordium. 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De forskellige bølger af hæmatopoietiske precursorceller overlapper delvist inden for en kort tidsramme, hvilket gør analysen af ​​bidraget fra hver bølge af udviklingshematopoiesis til immunceller teknisk teknisk udfordrende. Tamoxifen-inducerbare Cre-systemer giver mulighed for at mærke specifikke celler på en temporær inducerbar måde og udføre linieanalyse i embryoner eller voksne uden behov for ex vivo eller in vitro- kultur eller transplantation. I tamoxif...

Der er flere på hinanden følgende men overlappende bølger af hæmatopoietiske stamceller under udvikling, hvis myeloid afkom forbliver i voksenalderen. For det første kommer unipotente &quot;primitive&quot; forfædre frem i muskelblomme sac 1 , 2 mellem E7.5-E8.25 og give anledning til embryonale makrofager uden noget monocytisk mellemprodukt. Hvorvidt makrofager afledt af primitive stamceller fortsætter i den voksne hjerne, da microglia fortsat er et emne for aktiv undersøgelse. For det andet opstår Erythro-Myeloid Precursors (EMP&#39;er) i æggeblomme Sac ved E8.5, indtræder blodbanen og koloniserer embryoet. EMP&#39;er kommer frem fra æggeblomme sac hæmogent endotel i en Runx1-afhængig endotel-til-hæmatopoietisk overgang 3 , 4 . Mens EMP kan differentiere i makrofager inden for æggeblommehalsen, koloniserer de også føtalelever fra embryonal dag (E) 9 5 og adskiller sigTia i erythrocytter, megakaryocytter, makrofager, monocytter granulocytter og mastceller 6 . De makrofager, der stammer fra EMP&#39;er, udviser proliferativ kapacitet i udviklings- og voksenvæv. Hvorvidt EMP-afledte makrofager omgår monocytfasen af ​​differentiering er stadig kontroversiel, da meget lidt er kendt om deres differentieringsvej 7 , 8 . Endelig kommer hæmatopoietiske stamceller (HSCs) frem til E10.5 inden for det egentlige embryo fra aorta-gonad-mesonephros-regionen og migrere til føtalelever. HSC&#39;er med langvarig repopulationskapacitet registreres kun efter E11 (ved 42-somite-par-scenen) 9 . Der udvider og differentieres de fra E12.5, indtil definitiv hæmatopoiesis begynder at skifte til knoglemarv, som bliver det overvejende sted for blodcelleproduktion i postnatalt liv 10 . SpAtiel og tidsmæssig overlapning i fremkomsten såvel som delte immunfænotypiske markører har hidtil hæmmet vores evne til at skelne de specifikke bidrag fra disse bølger af embryonale hæmatopoietiske stamceller. Mens både EMP og HSC genereres på en Runx1-afhængig måde og udtrykker transkriptionsfaktoren Myb og vækstfaktorreceptoren Csf1r (kolonistimulerende faktor 1-receptor, også kendt som makrofagkoloniestimulerende faktorreceptor) blandt andre, kan EMP&#39;er skelnes fra HSCs ved deres mangel på lymfoide potentiale, både in vitro og in vivo , deres mangel på langsigtet repopuleringspotentiale og mangel på overfladekspression af linjemarkøren Sca- 11 . Genetiske skæbne kortlægningsmodeller er nødvendige for at karakterisere makrofag ontogeni, da de tillader målretning af embryonale forfædre på en celle-specifik og tidsspecifik måde. Her præsenterer vi skæbne-kortlægningsprotokollen anvendt i vores laboratorium for at diskriminere mellem de to linjerS af makrofager fundet i de fleste voksne væv: HSC-afledte infiltrerende makrofager og HSC-uafhængige residente makrofager. Tissue stationære makrofager er blevet sporet tilbage til Myb-uafhængige precursorceller udtrykker cytokinreceptor Csf1r 12 og er til stede i embryoet på E8.5-E10.5 anvendelse af tre komplementære skæbne-kortlægningsstrategier 6. For at studere æggeblomme sac hæmatopoiesis uden mærkning af føtal HSCs bruger vi en transgen stamme, Csf1r MeriCreMer , der udtrykker et tamoxifen-inducerbart fusionsprotein af &quot;forbedret&quot; Cre recombinase og to musestrogenreceptorer (Mer-iCre-Mer) under kontrol af Csf1r-promotoren. Derfor vil Cre recombinasen være aktiv i Csf1r-ekspresserende celler under et begrænset tidsvindue. Når det anvendes med en reporterstamme indeholdende et fluorescerende protein nedstrøms for en lox-STOP-lox-kassette (Rosa26 LSL-eYFP ), vil det føre til permanentEnt genetiske mærkning af cellerne til stede på tidspunktet for induktion men også af deres afkom. Administration ved E8.5 af aktiv form af tamoxifen, 4-hydroxytamoxifen (OH-TAM), EMP&#39;er og makrofager uden mærkning af æggeblomme sac unipotente &quot;primitive&quot; forfædre eller føtal HSCs. Dermed har vi karakteriseret immunophenotypen af ​​EMP&#39;er og deres afkom under embryonisk udvikling samt vurderet bidraget af æggeblomme-sac-afledte makrofager til de voksne makrofagpuljer. Yderligere arbejde er nødvendigt for at karakterisere, om primitive stamcellerafledte makrofager også mærkes med denne tilgang og om de kan bidrage til voksne makrofagpuljer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#5 Straight Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>MC19/B Pascheff-Wolff Spring Scissors&#160;</td>
			<td>Fine Science Tools</td>
			<td>15371-92</td>
			<td></td>
		</tr>
		<tr>
			<td>8.5 cm straight scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Kit-PE (clone 2B8)</td>
			<td>BD Pharmingen</td>
			<td>553355</td>
			<td>1/200 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45.2 APC-Cy7 (clone 104)</td>
			<td>Sony</td>
			<td>1149120</td>
			<td>1/100 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse AA4.1-APC (CD93, clone AA4.1)</td>
			<td>eBioscience</td>
			<td>17-5892</td>
			<td>1/100 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse Ter119 PerCP-Cy5.5 (clone Ter119)</td>
			<td>Biolegend</td>
			<td>560512</td>
			<td>1/200 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse F4/80-BV421 (clone BM8)</td>
			<td>BD Pharmingen</td>
			<td>123137</td>
			<td>1/100 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD11b PE-Cy7 (clone M1/70)</td>
			<td>BD Pharmingen</td>
			<td>552850</td>
			<td>1/200 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD16/CD32 (Mouse BD Fc Block, clone 2.4G2)</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Fischer Scientific</td>
			<td>12559069</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Fischer Scientific</td>
			<td>11570506</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D&#160;</td>
			<td>Roche</td>
			<td>11088882001</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>D4527-20KU</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Dutscher Dominique</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well tissue culture plate</td>
			<td>Dutscher Dominique</td>
			<td>353224</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plate</td>
			<td>Fischer Scientific</td>
			<td>11874235</td>
			<td></td>
		</tr>
		<tr>
			<td>96 wells U-shape bottom tissue culture plate</td>
			<td>Dutscher Dominique</td>
			<td>353227</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Syringe Plastipak 2 mL&#160;</td>
			<td>Dutscher Dominique</td>
			<td>300185</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon grid 100 &#181;m cell strainers</td>
			<td>Dutscher Dominique</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon grid 70 &#181;m cell strainers</td>
			<td>Dutscher Dominique</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Falcon tubes</td>
			<td>Dutscher Dominique</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup GP Millipore filtration kit, 0.2 &#956;m</td>
			<td>Dutscher Dominique</td>
			<td>51246</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>(Z)-4-HYDROXYTAMOXIFEN 25mg</td>
			<td>Sigma</td>
			<td>H7904-25MG</td>
			<td>Special care should be taken when preparing and working with tamoxifen and its derivates. Always use appropriate personal protective equipment</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P3972</td>
			<td>Always use appropriate personal protective equipment</td>
		</tr>
		<tr>
			<td>PEG-35 castor oil (Kolliphor/Cremophor EL)</td>
			<td>Sigma</td>
			<td>C5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Sunflower oil</td>
			<td>Sigma</td>
			<td>S5007-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Sigma</td>
			<td>24103-1L-R-D</td>
			<td>Always use appropriate personal protective equipment and use under the fumehood</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E9884-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI, 1 ML (1 MG/ML IN WATER)</td>
			<td>Fischer Scientific</td>
			<td>10116287</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX S B2-R3-V4-Y4 flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>B75408</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX Daily QC Fluorospheres</td>
			<td>Beckman Coulter&#160;</td>
			<td>B53230</td>
			<td></td>
		</tr>
		<tr>
			<td>VersaComp Antibody Capture Bead kit (2x5 mL)</td>
			<td>Beckman Coulter&#160;</td>
			<td>B22804</td>
			<td></td>
		</tr>
		<tr>
			<td>FVB-Tg(Csf1r-cre/Esr1*)1Jwp/J</td>
			<td>The Jackson laboratory</td>
			<td>19098</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J</td>
			<td>The Jackson laboratory</td>
			<td>6148</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of erythromyeloid progenitors and their progeny in the mouse embryo by flow cytometry

Makrofager er faglige fagocytter fra den medfødte arm af immunsystemet. I steady-state findes sessile makrofager i voksne væv, hvor de fungerer som frontlinjesangivelser af infektion og vævsskade. Mens andre immunceller kontinuerligt fornyes fra hæmatopoietiske stamceller og stamceller (HSPC), der er placeret i knoglemarven, har en række makrofager, der er kendt som residente makrofager, vist sig selvforsynet i væv uden input fra knoglemarvs HSPC&#39;er. Denne slægt er eksemplificeret ved microglia i hjernen, Kupffer celler i leveren og Langerhans celler i epidermis blandt andre. Tarm- og colon lamina propria er de eneste voksne væv uden HSPC-uafhængige bosiddende makrofager. Nylige undersøgelser har identificeret, at hjemmehørende makrofager stammer fra den ekstra-embryonale æggeblomme-hæmatopoiesis fra stamceller, der er forskellige fra føtal hæmatopoietiske stamceller (HSC). Blandt æggeblomme sac definitiv hæmatopoiesis, eRythromyeloid progenitorer (EMP) giver anledning til både erythroid og myeloidceller, især residente makrofager. EMP genereres kun i æggeblomme sac mellem E8.5 og E10.5 udviklingsdage, og de migrerer til føtalelever så tidligt som omsætning er forbundet, hvor de udvides og differentieres til mindst E16.5. Deres afkom omfatter erythrocytter, makrofager, neutrofiler og mastceller, men kun EMP-afledte makrofager fortsætter indtil voksenalderen i væv. Den forbigående karakter af EMP-fremkomsten og den tidsmæssige overlapning med HSC-generering gør analysen af ​​disse forgængere vanskelig. Vi har etableret en tamoxifen-inducerbar skæbneplanlægningsprotokol baseret på ekspression af makrofag-cytokinreceptor Csf1r-promotoren til karakterisering af EMP- og EMP-afledte celler in vivo ved flowcytometri.

Genetisk skæbne kortlægning blev opnået ved administration ved E8.5 af OH-TAM til Csf1r-Mer-iCre-Mer kvinder med parret med en Rosa26-LSL-eYFP reporter. I nærvær af OH-TAM fører udskæring af stopkassetten til den permanente ekspression af YFP i cellerne der udtrykker Csf1r . Vi samler to hæmatopoietiske væv: æggeblomme og fostrelever og et ikke-hæmatopoiet væv, neuroektoderm, fra embryonerne ved E10.5. Encellesuspensioner blev opnået ved enzymatisk og mekan...

Watch this Scientific Journal Video about Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry at JoVE.com

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Selv om infiltrerende makrofager rekrutteres kontinuerligt til voksne væv fra cirkulerende forstadier, frøer residente makrofager deres væv under udvikling, hvor de opretholdes uden yderligere indløb fra forfædre. Forfædrene for residente makrofager blev for nylig identificeret. Her præsenterer vi metoder til den genetiske skæbneplanlægning af de residente makrofagprogenitorer.

Identifikation af erythromyeloidprogenitorer og deres afkom i musembryoen ved flowcytometri

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Research

JoVE Journal

Developmental Biology

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Karakterisering af tymisk Bundfældning Progenitorer i Mouse Embryo Brug<em&gt; In vivo</em&gt; Og<em&gt; In vitro</em&gt; Analyser

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Ex Vivo Kultur Faryngeal Arches at studere hjerte og Muskel Progenitorer og deres niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

Trin-specifik Sortering af Mouse spermatider ved flowcytometri

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

Isolering og analysere celler i bugspytkirtlen mesenchym ved flowcytometri

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

En standardiseret tilgang til multispecies Rensning af mammale mannlige germeller ved hjælp af mekanisk vævsdissociation og flowcytometri

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Bioengineering af humaniserede knoglemarv mikro miljøer i mus og deres visualisering ved Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Shifting zebrafisk dødbringende skelet Mutant penetrans af afkom test

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation af stillads-fri, tre-dimensionelle Insulin udtrykker Pancreatoids fra mus i bugspytkirtlen stamfaderen In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Brug af hæmatopoietisk stamcelletransplantation at vurdere oprindelsen af myelodysplastisk syndrom

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Realtids Live-celle flowcytometri at undersøge Calcium tilstrømning, Pore dannelse og fagocytose af P2X7 receptorer i voksne neurale stamceller

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...