Name: assessing the development of murine plasmacytoid dendritic cells in peyer's patches using adoptive transfer of hematopoietic progenitors
Uploaded: 2014-03-17T16:30:00
Description: Watch this Scientific Journal Video about Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors at JoVE.com

Vi takker Drs. Alex Gelbard og Willem Overwijk for rådgivning om hydrodynamisk genoverførsel. Dette arbejde blev støttet af tilskud fra NIH (AI098099, SSW), MD Anderson Cancer Center for Epigenetik, MD Anderson Center for Inflammation og Kræft (SSW) og RE Bob Smith Education Fund (HSL).

Institutionel godkendelse skal indhentes på forhånd for alle eksperimentelle manipulationer beskrevet heri involverer mus. Disse omfatter anvendelsen af C57BL6-mus til isolering af knoglemarvprogenitorceller, IFNAR - / - mus som modtagere for adoptiv overførsel af hæmatopoietiske progenitorer og anvendelse af HGT for cytokin overekspression in vivo. Passende bolig og pasning af dyr skal også gives af investigator eller institution. Desuden kan der kræves institutionelle godkendelse for...

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Adoptiv transfer teknik beskrevet heri vurderede bidrag CDPs til PP pDC befolkning i modtagerlandene mus, der er mangelfuld i PP pdCs (f.eks IFNAR - / - mus). I fremtidige eksperimenter, vil det være vigtigt at vurdere potentialet af andre progenitordelmængder generere PP pdCs, især hvorvidt PP pdCs stammer fra Flt3 + CLPS. Dette spørgsmål er vigtigt, fordi det er uklart, hvorfor PP pdCs er unikt følsomme over for IFNAR-STAT1 signaler for PP periodisering 5, og hvis PP pdC...

Her viser vi en protokol at vurdere, om de fælles dendritiske-stamceller (CDPs) er i stand til at give anledning til plasmacytoid dendritiske celle (PDC) befolkning Peyers patch (PP). Det overordnede mål om at anvende denne metode var at evaluere den udviklingsmæssige regulering af pdCs i Peyer patch (PP pdCs). Grunden til dette er vigtigt, er, at PP pdCs afviger fra pdCs findes i andre væv, herunder knoglemarv, blod og milt, og det er derfor uklart, om PP pdCs og andre PDC populationer er udviklingsmæssigt og / eller funktionelt beslægtede. Konkret er pdCs almindeligt kendt for at være den vigtigste type I interferon (IFN) producenter inden for det hæmatopoietiske system, reagerer på Toll-lignende receptor 7 og 9 (TLR7 / 9) stimulation ved hurtig IFN-sekretion 1-3. Men PP pdCs er mangelfuld i produktion af type I IFN reaktion på TLR agoniststimulering 4,5. Desuden PP pdCs adskiller sig også fra pdCs findes i knoglemarv og milt ikræver signaler fra type I interferon (IFN)-receptor (IFNAR1) eller IFN signalering molekyle STAT1 for deres udvikling og / eller akkumulering 5. Disse data har foreslået muligheden for, at forskellige reguleringsmekanismer styrer PP pdCs versus pdCs i andre organer (f.eks knoglemarv, milt) 5. Det rationale, der førte til udviklingen af ​​denne fremgangsmåde var baseret på de seneste fremskridt i forståelsen dendritisk celle (DC) biologi. De fleste, hvis ikke alle, DC delmængder stammer fra hæmatopoietiske stamceller, der udtrykker FMS-lignende tyrosinkinase 3 receptor (Flt3) 6-10, men er DC udvikling ikke begrænset til klassiske myeloide og lymfoide veje. For eksempel FLT3 + fælles myeloide stamceller (CMPs, lin - IL-7R - Sca-1 - c-kit + CD34 + FcyR lo / -) giver anledning til CDPs (lin - c-kit lo CD115 + Flt3 +), which yderligere differentiering i pdCs og konventionelle udviklingslande (CDCS) 9,10. Derimod Flt3 + fælles lymfoide progenitorer (CLPS Lin - IL-7R + Sca-1 lo c-kit lo) udvikle primært i pdCs 11. Derfor forudgående undersøgelser tyder pdCs opstå fra mindst 2 forskellige hæmatopoietiske befolkninger under reguleringen af ​​Flt3L selvom den typiske analyse er begrænset til knoglemarven, milten og / eller blod PDC delmængder. Således progenitorpopulationen (r), der genererer PP pdCs krævede undersøgelse. Forståelse oprindelsen af ​​PP pdCs vil kaste lys over, om de deler fælles udviklingsmæssige veje med andre PDC befolkninger, eller benytte forskellige mekanismer i løbet af deres generation i PP. En unik fordel af den fremgangsmåde, der er beskrevet heri, er brugen af ​​mus, der viser en alvorlig mangel i PP pdCs som modtagere for adoptiv overførsel af hæmatopoietiske stamceller. Mus med genettic deletion i genet, der koder IFNAR1 (IFNAR - / - mus) og STAT1 (Stat1 - / -) afslørede en slående depletering i PP pdCs 5. Derfor disse stammer giver et miljø, hvor PP pdCs er reduceret, så adoptivforældre undersøgelser transfer, der skal udføres i fravær af potente celle ablation regimer som dødelig bestråling. En yderligere styrke metode præsenteres her, er brugen af ​​hydrodynamiske genoverførsel (HGT) at stimulere forhøjede cirkulerende mængder af Flt3L. Dette tilvejebringer en omkostningseffektiv metode til at inducere Flt3L in vivo over for injektion af rekombinant protein. Talrige undersøgelser, herunder dem i vores laboratorium, har ansat HGT at fremkalde cytokin mængder i en række forskellige eksperimentelle betingelser 5,12,13. Arbejdsdelingen og præcise immunfunktionerne for udviklingslandene er af stor interesse i immunologi. Navnlig pdCs er vigtige mediatorer af oral tolerance og systemisk anti-viralreaktioner, men de synes også at bidrage til udviklingen og persistens af autoimmunitet og cancer 14-17. Den heri beskrevne protokol vil give udviklingsmæssige mekanismer, der regulerer PP pdCs at være mere fuldt udforsket. Desuden kan denne tilgang kunne undersøgelser for at vurdere PP PDC-funktionen, og kan udvides til at forstå regulering og funktion af andre immun populationer i PPs.

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			<td height="21" style="height:21px;width:260px;">C57BL/6J</td>
			<td style="width:201px;">JAX</td>
			<td style="width:177px;">664</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">B6.SJL</td>
			<td style="width:201px;">JAX</td>
			<td style="width:177px;">2014</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RPMI</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:177px;">11875-093</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">15 ml conical tubes</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352095</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">50 ml conical tubes</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352070</td>
			<td style="width:229px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sterile surgical tweezers</td>
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			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sterile small pair scissors</td>
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			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
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			<td height="21" style="height:21px;width:260px;">Sterile large pair scissors</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
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			<td height="21" style="height:21px;width:260px;">40 &#956;m cell strainer</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352340</td>
			<td style="width:229px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">35 &#956;m cell strainer cap tubes</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352235</td>
			<td style="width:229px;"></td>
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			<td height="21" style="height:21px;width:260px;">RBC lysing buffer</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:177px;">R7757</td>
			<td style="width:229px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:260px;">FACS buffer</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;">PBS, 2 mM EDTA, 1% FCS, filter sterilized</td>
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			<td height="21" style="height:21px;width:260px;">Percoll</td>
			<td style="width:201px;">GE Healthcare</td>
			<td style="width:177px;">17089102</td>
			<td style="width:229px;"></td>
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			<td height="21" style="height:21px;width:260px;">10XHBSS</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:177px;">H4641</td>
			<td style="width:229px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Collagenase IV</td>
			<td style="width:201px;">Worthington</td>
			<td style="width:177px;">LS004188</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Goat ant-rat IgG microbeads</td>
			<td style="width:201px;">Milteyni Biotec</td>
			<td style="width:177px;">130-048-501</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">LD column</td>
			<td style="width:201px;">Milteyni Biotec</td>
			<td style="width:177px;">130-042-901</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-D3</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">555273</td>
			<td style="width:229px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-CD19</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553783</td>
			<td style="width:229px;"></td>
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			<td height="21" style="height:21px;width:260px;">Rat anti-CD11b</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553308</td>
			<td style="width:229px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-CD11c</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553799</td>
			<td style="width:229px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-Ter119</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553671</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD3 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0031</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD19 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0193</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD11b (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0112</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD11c (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0114</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-F4/80 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-4801</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Ter119 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-5921</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Sca-1 (PE.Cy7)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">25-5981</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD115 (APC)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">17-1152</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-c-kit (APC.Cy7)&#160;</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">47-1171</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-IL-7R (Pacific Blue)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">48-1271</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Flt3 (PE)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">12-1351</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD45.1 (APC.Cy7)</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">560579</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD45.2 (PE.Cy7)</td>
			<td style="width:201px;">Biolegend</td>
			<td style="width:177px;">109830</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD11c (Pacific Blue)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">48-0114</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-B220 (APC)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">25-0452</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Siglec-H (PE)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">12-0333</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-PDCA-1 (FITC)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">Nov-72</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Cell sorter</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;"></td>
			<td style="width:229px;">e.g. BD Fortessa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Heat lamp</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Mouse restrainer</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">1 ml syringes</td>
			<td style="width:201px;">Becton Dickinson</td>
			<td style="width:177px;">309602</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">27&#189; G needles (sterile)</td>
			<td style="width:201px;">Becton Dickinson</td>
			<td style="width:177px;">305109</td>
			<td style="width:229px;"></td>
		</tr>
	</tbody>
</table>

assessing the development of murine plasmacytoid dendritic cells in peyer's patches using adoptive transfer of hematopoietic progenitors

Denne protokol beskriver en metode til at analysere evnen af ​​oprensede hæmatopoietiske progenitorer til at generere plasmacytoid dendritiske celler (PDC) i tarm Peyerske plaques (PP). Fælles dendritiske-stamceller (CDPs Lin - c-kit lo CD115 + Flt3 +) blev oprenset fra knoglemarven hos C57BL6 mus ved FACS og overføres til recipientmus, der mangler en væsentlig pDC befolkning i PP, i dette tilfælde, IFNAR&#39;en - / - mus blev anvendt som overførselsmodtagere. I nogle mus blev overekspression af dendritiske cellevækstfaktor Flt3-ligand (Flt3L) fuldbyrdet før adoptiv overførsel af CDPs, ved hjælp af hydrodynamisk genoverførsel (HGT) af Flt3L-kodende plasmid. Flt3L overekspression udvider DC-populationer stammer fra overført (eller endogene) hæmatopoietiske stamceller. På 7-10 dage efter stamfader overførsel blev pdCs, der opstår fra de adoptivt overførte stamfædre skelnes fra modtagerceller pågrundlag af CD45 markør udtryk, med pdCs fra overførte CDPs være CD45.1 + og modtagere bliver CD45.2 +. Evnen af ​​overførte CDPs at bidrage til pDC befolkning i PP og reagere på Flt3L blev vurderet ved flowcytometri PP enkelte cellesuspensioner fra recipientmus. Denne fremgangsmåde kan anvendes til at teste, om andre progenitorpopulationer er i stand til at generere PP PDCs. Desuden kan denne fremgangsmåde anvendes til at undersøge den rolle, de faktorer, der forudsiges at påvirke pDC udvikling i PP, ved at overføre progenitordelmængder med en passende knockdown, knockout eller overekspression af det formodede udviklingsmæssige faktor og / eller ved at manipulere cirkulerende cytokiner via HGT . Denne metode kan også tillade analyse af, hvordan PP pdCs påvirker hyppigheden eller funktion af andre immun delmængder i KKS. En unik egenskab ved denne fremgangsmåde er anvendelsen af IFNAR - / - mus, som viser alvorligt reducerede PP pdCs forhold til vildtype dyr, derfor allowing rekonstituering af PP pdCs i fravær af forstyrrende effekter fra dødelig bestråling.

Vores resultater viser gating strategi til isolering af CDPs fra muse-knoglemarvsceller (Figur 1), som beskrevet i protokol 2 og 3. CDPs omfatter cirka 0,1% af den samlede knoglemarvsceller, og omkring 4-6 x 10 4 CDPs kan isoleres fra en mus. Ved adoptiv overførsel, CDPs differentierer til pdCs og CDCS 10. <img alt="Figur 1" fo:content-width="5in" fo:src="/files/ftp_upload/51189/51189fig1highres.jpg" src="/files/ftp...

Watch this Scientific Journal Video about Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors at JoVE.com

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

Denne protokol beskriver eksperimentelle procedurer til at vurdere differentiering af plasmacytoid dendritiske celler i Peyer patch fra fælles dendritiske celle stamceller ved hjælp af teknikker, der involverer FACS-medieret celle isolation, hydrodynamisk genoverførsel, og flow analyse af immun delmængder i Peyers patch.

Vurdere udviklingen i murine Plasmacytoid dendritiske celler i Peyer&#39;ske Patches Brug adoptiv overførsel af Hematopoietic Progenitorer

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

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Isolering og immunfarvning Lymfocytter og dendritiske celler fra murine Peyerske plaks

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Vurdering af Innate Sensing af HIV-1 inficerede CD4<sup&gt; +</sup&gt; T-celler ved plasmacytoide dendritiske celler ved hjælp af en<em&gt; Ex vivo</em&gt; Co-dyrkningssystem.

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

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In vitro Generation af murint Plasmacytoide dendritiske celler fra fælles Lymfoid progenitorer ved hjælp af 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 ...

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

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