Name: in vitro generation of murine plasmacytoid dendritic cells from common lymphoid progenitors using the ac-6 feeder system
Uploaded: 2015-11-23T17:00:00
Description: Watch this Scientific Journal Video about In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System at JoVE.com

Vi er takknemlige for legene. Markus Manz og Irving Weissman for å gi reagenser. Vi erkjenner også tjenesten leveres av Flowcytometriske Analyse og Cell Sorting Kjerne Facility av første og andre Kjernelaboratoriet ved National Taiwan University College of Medicine og NTU sykehus, henholdsvis. Dette arbeidet ble støttet av departementet for vitenskap og teknologi, Taiwan (MEST 102-2320-B-002-030-My3) og National Health Research Institutes, Taiwan (NHRI-EX102-10256SI og NHRI-EX103-10256SI).

C57BL / 6 villtype mus ble kjøpt fra National Laboratory Animal Senter (NLAC), Taiwan. Alle mus ble avlet og holdt under patogenfrie forhold. Protokoller og dyr bruk prosedyrer ble gjennomgått og godkjent av Institutional Animal Care og bruk komité National Taiwan University College of Medicine (Permit Number: 20120075). I tillegg forskere har gjort vårt ytterste for å redusere potensialet for smerte, lidelse, eller nød i dyrene mens du utfører eksperimenter. Alle prosedyrer beskrevet ble utført ved RT med hansker. <p class="jov...

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Her beskriver vi et in vitro kultursystem for robust generering av DC, og PDCs spesielt fra et lite antall CLPs. Det unike med dette kultursystem er på grunn av bruk av AC-6-celler, en cellelinje stromal, som forer. Denne metoden har vist seg å tilveiebringe ikke bare cytokiner, slik som IL-7, SCF, M-CSF, og FL 20, men også den celle-celle-kontakt 21 er nødvendig for å understøtte utvikling DC. AC-6 celler har blitt brukt tidligere for å lette studiet av in vitro DC utvikli...

Dendrittiske celler (DCS) er profesjonell antigen-presenterende celler som spiller en viktig rolle i å kontrollere en immunresponser. Mens DC er heterogene, de kan grovt deles inn i to populasjoner, konvensjonell DC (CDCS) og plasmacytoid DC (PDCs) 2,3. I tillegg til lymfoide organer, er CDCS og PDCs også funnet i vev innbefattende lunge, tarm, hud og 2. Morfologi CDCS og PDCs forskjellig, med CDCS utviser dendrite-lignende anslag og formen på PDCs blir mer plasma celle-lignende. I tillegg er det vanlig mus DC markør, CD11c, mer sterkt uttrykt på CDCS enn på PDCs. Videre kan CDCS videre deles inn CD11b + CD24 - CDCs og CD11b - CD24 + CDCS, som begge uttrykker høyere nivåer av MHC klasse II og samtidig stimulerende molekyler enn gjøre PDCs 2. Modne PDCs, på den annen side, har vist seg selektivt å uttrykke Siglec-H og BST2 4. Functionally, CDCS er bedre antigenpresenterende celler enn er PDCs; men kan PDCs produsere en enorm mengde av type I interferon på virus infeksjon eller inflammatorisk stimulering 5. Begge CDCS og PDCs er kortvarige, og derfor må stadig etterfylles fra stamfedre i benmargen (BM) 6,7. Adoptiv overføring av felles myeloide stamceller (CMPS) og vanlige lymfoide progenitors (CLPs) inn lethally-bestrålte mus viser at CDCS og PDCs kan genereres fra begge linjene 8-10. Imidlertid er det en undergruppe av PDCs som uttrykker RAG1 / 2 og besitter rearrangerte DJ segmenter ved avIgH locus 11,12. Disse cellene deler også molekylære likheter med B-lymfocytter, inkludert uttrykk for B220 markør, nukleinsyre-sensing reseptorer (TLR7 / TLR9), og transkripsjonsfaktorer (SPIB og Bcl11a) 13, har alle antas å være underskrifter fra lymfoidlinjen. Derfor CLPs kan være gode valg for in vitro generasjon PDCs grunn av avstamning likheten. Mens frekvensen av både CDCS og PDCs i mennesker og mus er meget lav 6, DC, særlig CDCS, kan genereres in vitro fra BM eller progenitorceller i nærvær av cytokiner, slik som GM-CSF 11,14 eller Flt3-ligand (FL ) bruker mater frie systemer 11,15,16. Dessverre er det imidlertid ikke mulig å fremstille et stort antall PDCs in vitro ved hjelp av FL 11,15,16. Tidligere har vi vist at PDCs effektivt kan genereres in vitro fra CLPs bruker AC-6 mater system 17. Fordelen med å bruke en AC-6 stromal cellelinjen i kultursystemet er at det gir celle-celle-kontakt og sekresjon av cytokiner som støtter generering av et stort antall PDCs fra CLPs. Selv om produksjonen med dette systemet er ganske robust, er forsiktig replikering av prosedyrene som er beskrevet nedenfor kreves in For å sikre effektiv generasjon PDCs.

<table>
	<tbody>
		<tr>
			<td>Anti-mouse Ly6g/Ly6c (PE), clone RB6-8C5</td>
			<td>Biolegend</td>
			<td>108408</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse NK1.1 (PE), clone PK136</td>
			<td>Biolegend</td>
			<td>108708</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse&#160; CD11b (PE), cloneM1/70</td>
			<td>Biolegend</td>
			<td>101208</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse CD19 (PE), clone eBio1D3</td>
			<td>Biolegend</td>
			<td>115508</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse B220 (PE), clone RA3-6B2</td>
			<td>Biolegend</td>
			<td>103208</td>
			<td>linage marker/FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD3 (PE), clone 17A2</td>
			<td>Biolegend</td>
			<td>100308</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse CD8a (PE), clone 53-6.7</td>
			<td>Biolegend</td>
			<td>100707</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse MHC-II (PE), clone NIMR-4</td>
			<td>Biolegend</td>
			<td>107608</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse Ter119 (PE), clone TER-119</td>
			<td>Biolegend</td>
			<td>116208</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse Thy1.1 (PE), clone HIS51</td>
			<td>eBioscience</td>
			<td>12-0900-83</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse M-CSFR (APC), clone AFS98</td>
			<td>Biolegend</td>
			<td>135510</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse c-Kit (PerCP/Cy5.5), clone 2B8</td>
			<td>Biolegend</td>
			<td>105824</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse Sca-1 (FITC), clone D7</td>
			<td>Biolegend</td>
			<td>108106</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse IL-7Ra (PE/Cy7), clone A7R34</td>
			<td>Biolegend</td>
			<td>135014</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c (PerCP/Cy5.5), clone N418</td>
			<td>Biolegend</td>
			<td>117328</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b (FITC), clone M1/70</td>
			<td>Biolegend</td>
			<td>101206</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Cell sorter</td>
		</tr>
		<tr>
			<td>FACS sorting tube&#160;</td>
			<td>BD Biosciences</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>FlowJo LLC</td>
			<td></td>
			<td>Flow analysis sofrware</td>
		</tr>
	</tbody>
</table>

in vitro generation of murine plasmacytoid dendritic cells from common lymphoid progenitors using the ac-6 feeder system

Plasmacytoid dendrittiske celler (PDCs) er kraftig type I interferon (IFN-I) produserende celler som aktiveres som respons på infeksjon eller ved inflammatoriske responser. Dessverre studie av PDC-funksjonen er hindret av deres lave frekvensen i lymfoide organer, og eksisterende metoder for in vitro DC generasjon overveiende favorisere produksjon av CDCs løpet PDCs. Her presenterer vi en unik tilnærming til effektivt å generere PDCs fra vanlige lymfoide stamfedre (CLPs) in vitro. Nærmere bestemt protokollen beskrevet detaljer hvordan man skal rense CLPs fra benmarg og generere PDCs ved coculturing med y-bestrålte AC-6 feeder-celler i nærvær av Flt3-ligand. En unik egenskap ved denne kulturen systemet er at CLPs migrere under AC-6 celler og bli brosteins området dannende celler, et avgjørende skritt for å utvide PDCs. Morfologisk forskjellige DC, nemlig PDCs og CDCS, ble generert etter ca 2 uker med en sammensetning av 70-90% PDCs under optimale forhold. Vanligvis er antallet PDCs som genereres ved denne fremgangsmåten omtrent 100 ganger av antall CLPs seeded. Derfor er det et nytt system som brukes til å generere robuste store antall PDCs som kreves for å legge til rette for ytterligere studier, og i utvikling og funksjon av disse cellene.

Totalt 7 BM 4-6x10 celler blir vanligvis isolert fra lårben og skinneben over en 6-8 ukers gamle, villtype C57BL / 6 mus. Å sortere ut CLPs er samlede BM celler farget med PE-konjugerte antistoffer mot avstamning markører (CD3, CD8, B220, CD19, CD11b, Gr-1, Thy1.1, NK1.1, TER119, og MHC-II), anti c-kit-PerCP / Cy5.5, anti-Sca-1-FITC, anti-M-CSFR-APC og anti-IL-7Rα-PE / Cy7, analyseres og sorteres med en celle-sorterer. Sorterings strategi for CLP er vist i figur 1. Vanlig...

Watch this Scientific Journal Video about In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System at JoVE.com

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System (Video) | JoVE

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

In Vitro Generering av murine Plasmacytoid dendrittiske celler fra Common Lymfoid stamfedre bruker AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

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Immunology and Infection

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

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

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Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors (Video) | JoVE

Vurdere utvikling av Murint Plasmacytoid dendrittiske celler i Peyer sin Patches Bruke Adoptive Overføring av Hematopoietic stamfedre

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System. (Video) | JoVE

Vurdere Medfødt Sensing av HIV-1 infiserte CD4<sup&gt; +</sup&gt; T-celler ved Plasmacytoid dendrittiske celler ved hjelp av en<em&gt; Ex vivo</em&gt; Co-kultursystem.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes (Video) | JoVE

Generering av umoden, moden og tolerogene dendrittiske celler med ulik Metabolsk fenotyper

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

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

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow (Video) | JoVE

Fluorescens-aktivert cellesortering for Rensing av Plasmacytoid dendrittiske celler fra mus Bone Marrow

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

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood (Video) | JoVE

Generering av menneske moncyttavledede dendrittiske celler fra Whole Blood

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

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro (Video) | JoVE

En roman mater-fritt System for masseproduksjon av Murine naturlige drepe celler In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...