Name: isolation of dendritic cells from the human female reproductive tract for phenotypical and functional studies
Uploaded: 2018-03-13T15:00:00
Description: Watch this Scientific Journal Video about Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies at JoVE.com

Støttet af NIH stipendier AI102838 og AI117739 (CRW). Vi takker Richard Rossoll for teknisk bistand. Flow cytometric analyse blev udført i DartLab, den delte ressource på Dartmouthsupported af (P30CA023108-37) og (P30GM103415-15).

Undersøgelser, hvor forsøgspersoner blev gennemført efter principperne udtrykt i Helsinki-erklæringen. Undersøgelser blev godkendt af Udvalget for beskyttelse af menneskelige emner (CPHS) og Dartmouth College institutionelle Review Board. Skriftligt informeret samtykke blev opnået før operationen fra HIV-negative kvinder, der gennemgår hysterektomier på Dartmouth-Hitchcock Medical Center (Libanon, NH). Uddannet patologer valgt vævsprøver fra EM, CX og ECX, gratis af patologiske læsioner og fjernt fra steder i...

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	<li>Wira, C. R., Rodriguez-Garcia, M., Patel, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+sex+hormones+in+immune+protection+of+the+female+reproductive+tract.">The role of sex hormones in immune protection of the female reproductive tract.</a> Nat Rev Immunol. 15 (4), 217-230 (2015).</li><li>Banchereau, J., Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+the+control+of+immunity.">Dendritic cells and the control of immunity.</a> Nature. 392 (6673), 245-252 (1998).</li><li>Schlitzer, A., McGovern, N., Ginhoux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+monocyte-derived+cells:+Two+complementary+and+integrated+functional+systems.">Dendritic cells and monocyte-derived cells: Two complementary and integrated functional systems.</a> Semin Cell Dev Biol. 41, 9-22 (2015).</li><li>Tagliani, E., Erlebacher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+function+at+the+maternal-fetal+interface.">Dendritic cell function at the maternal-fetal interface.</a> Expert Rev Clin Immunol. 7 (5), 593-602 (2011).</li><li>Kaldensjo, 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=Detection+of+intraepithelial+and+stromal+Langerin+and+CCR5+positive+cells+in+the+human+endometrium:+potential+targets+for+HIV+infection.">Detection of intraepithelial and stromal Langerin and CCR5 positive cells in the human endometrium: potential targets for HIV infection.</a> PLoS One. 6 (6), e21344 (2011).</li><li>Schulke, L., Manconi, F., Markham, R., Fraser, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+dendritic+cell+populations+during+the+normal+menstrual+cycle.">Endometrial dendritic cell populations during the normal menstrual cycle.</a> Hum Reprod. 23 (7), 1574-1580 (2008).</li><li>Ballweber, L., 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=Vaginal+langerhans+cells+nonproductively+transporting+HIV-1+mediate+infection+of+T+cells.">Vaginal langerhans cells nonproductively transporting HIV-1 mediate infection of T cells.</a> J Virol. 85 (24), 13443-13447 (2011).</li><li>Hladik, 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=Initial+events+in+establishing+vaginal+entry+and+infection+by+human+immunodeficiency+virus+type-1.">Initial events in establishing vaginal entry and infection by human immunodeficiency virus type-1.</a> Immunity. 26 (2), 257-270 (2007).</li><li>Duluc, D., 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=Functional+diversity+of+human+vaginal+APC+subsets+in+directing+T-cell+responses.">Functional diversity of human vaginal APC subsets in directing T-cell responses.</a> Mucosal Immunol. 6 (3), 626-638 (2013).</li><li>Herzenberg, L. 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Slimhinde DCs er en sjælden celle population stærkt påvirket af væv miljø, som ændrer deres fænotype og funktion, når de træder væv3. Mens blod afledt DCs er en meget nyttig model, de repræsenterer ikke fuldt ud mangfoldigheden af DC populationer fundet i væv. Derfor, for at forstå de unikke egenskaber af slimhinde DCs, dyrkning af primære celler fra væv er nødvendigt. Isolering af DCs fra forskellige anatomiske steder i FRT tilbyder mulighed for at studere DC funktion i vitro<...

FRT har den dobbelte funktion at beskytte mod patogener knappanel implantation og graviditet1. For at opnå dette, er FRT opdelte, med hver anatomiske region viser unikke histologiske, immunologiske og funktionelle karakteristika1.
DCs stede ved slimhindeinfektioner gøre kontakt med mikrober i lungen, tarmen og kønsorganerne, og give immun overvågning for potentielle patogener2. DCs har den unikke evne til at prime naive T-celler og udløse adaptive immunrespons3. DCs i FRT er også specialiseret til at tolerere fremmede antigener, som dem der findes i sæd og det udviklende foster, at vellykket graviditet4. Derfor, afhængigt af placeringen, DC fænotype og funktion vil være adskilte. Det er kendt at DCs er stærkt påvirket af væv miljø, således at deres antal, fænotype og funktion er ændret af væv miljø, hvor de er placeret3. Derfor, for at forstå den rolle, FRT DCs spille i smitsomme sygdomme, graviditet og kræft i FRT, bosiddende DCs skal studeres, da blod afledte DC modeller er utilstrækkelige til at løse de regulerende kompleksiteter fundet i FRT væv.
Karakterisering af humant væv bosiddende DCs udfordrende på grund af det lave antal celler i slimhinden væv og det er vanskeligt at opnå menneskelige vævsprøver. DCs er blevet undersøgt i FRT ved hjælp af Immunhistokemi5,6, som informerer om celleplacering inden for væv, men udelukker funktionelle studier og er begrænset af antallet af at identificere celle markører, der kan analyseres på én gang. Desuden har enkelt celle isolation protokoller for analysen af cytometric været udviklede7,8,9. Nogle af disse protokoller drage fordel af DCs vandrende evne til at isolere de celler, der overflyttes. Disse metoder normalt kræver overnight inkubationer og udvalg af aktiverede DCs, men tillader ikke for undersøgelse af DCs i steady state.
Her, ved hjælp af hysterektomi prøver, vi optimeret en protokol for at isolere DCs fra forskellige anatomiske steder i FRT, endometriet (EM), endocervix (CX) og ectocervix (ECX), der giver mulighed for både phenotypical og funktionelle analyser. Ved hjælp af en ikke-proteolytiske enzymatisk fordøjelsen protokol, kan vi fortsætte umiddelbart efter væv fordøjelse til celle isolation og flow cytometric karakterisering uden celle aktivering. Ved hjælp af flerfarvet flowcytometri og tilpasse funktionelle studier for lavt antal celler, kan vi identificere og karakterisere sjældne delmængder af DCs i de forskellige steder i FRT.

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			<td height="21" style="height:21px;width:273px;">Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td style="width:203px;">Hyclone</td>
			<td style="width:119px;">SH30015.03</td>
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			<td height="21" style="height:21px;">HEPES (1M)</td>
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			<td height="21" style="height:21px;">Collagenase IV</td>
			<td>Sigma</td>
			<td>C5138</td>
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			<td height="21" style="height:21px;">Deoxyribonuclease I</td>
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			<td height="21" style="height:21px;">D-glucose</td>
			<td>Sigma Aldritch</td>
			<td>50-99-7</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">0.22 um Stericup 500 mL&#160; filter</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">100mm x 15mm polystyrene petri dish</td>
			<td>Fisherbrand</td>
			<td>FB0875712</td>
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		<tr height="21">
			<td height="21" style="height:21px;">150mm x 15mm polystyrene petri dish</td>
			<td>Fisherbrand</td>
			<td>FB0875714</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">150mm x 25mm polystyrene dish</td>
			<td>Corning</td>
			<td>430599</td>
			<td>Treated cell culture dish</td>
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		<tr height="25">
			<td height="25" style="height:25px;">Isotemp Incubator</td>
			<td>Fisher Scientific</td>
			<td>FICO3500TABB</td>
			<td>5.0% CO2</td>
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		<tr height="21">
			<td height="21" style="height:21px;">American Rotator V</td>
			<td>American DADE</td>
			<td>R4140</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">250 um nylon mesh</td>
			<td>Sefar</td>
			<td>03-250/50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 um nylon mesh</td>
			<td>Sefar</td>
			<td>03-20/14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Beckman GS-6R Centrifuge</td>
			<td>Beckman</td>
			<td>358702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-VIVO 15 with Gentamicin L-Gln, Phenol Red, 1 L</td>
			<td>Lonza</td>
			<td>04-418Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human AB Serum</td>
			<td>Valley Biomedical</td>
			<td>HP1022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histopaque-1077</td>
			<td>Sigma Aldritch</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Solution (PBS)</td>
			<td>National Diagnostics</td>
			<td>CL-253</td>
			<td>pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dead Cell Removal Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pre-separation filter (30um)</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LS column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quadro MACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS multi-stand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>USB</td>
			<td>15694</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD1a Microbeads, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-051-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD14 Microbeads, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-050-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eFluor 670 cell proliferation dye</td>
			<td>eBiosciences</td>
			<td>65-0840-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well round bottom plate</td>
			<td>Falcon</td>
			<td>9/8/2866</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zombie yellow viability dye</td>
			<td>Biolegend</td>
			<td>423104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD3 APC/Cy7, anti-human</td>
			<td>Tonbo Biosciences</td>
			<td>25-0038-T100</td>
			<td></td>
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			<td height="21" style="height:21px;">CD4 PE, anti-human</td>
			<td>eBiosciences</td>
			<td>12-0048-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD8a FITC, anti-human</td>
			<td>Tonbo Biosciences</td>
			<td>35-0086-T100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gallios Flow Cytometer</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>B43618</td>
			<td>10 color, VBR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACSquant Analyzer 10</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-343</td>
			<td>8 color, VBR</td>
		</tr>
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isolation of dendritic cells from the human female reproductive tract for phenotypical and functional studies

Karakterisering af de menneskelige dendritiske celler (DCs) bopæl i slimhinden væv er udfordrende på grund af vanskeligheden ved at få udleveret prøver, og det lave antal DCs præsentere pr. væv. Men fænotype og funktion af DCs er modificeret af væv miljø, det er nødvendigt at analysere væv bosiddende DC populationer, da blodet stammer DCs ufuldstændigt afspejler kompleksiteten af DCs i væv. Her præsenterer vi en protokol for at isolere DCs fra menneskelige kvindelige forplantningsorganerne (FRT) ved hjælp af hysterektomi prøver der giver mulighed for både phenotypical og funktionelle analyser. Protokollen består af væv fordøjelse til at generere en enkelt celle blandet cellesuspension, efterfulgt af positiv magnetisk bead udvalg. Vores væv fordøjelsen protokol ikke spaltes celleoverflademarkører, som giver mulighed for phenotypical og funktionel analyse af DCs i steady state, uden natten inkubation eller celle aktivering. Denne protokol kan tilpasses til isolation af andre immun celletyper eller isolation af DCs fra andre væv.

Følgende væv fordøjelse, frigivelse af epitel ark og kirtler kan observeres, som vist i figur 1A; Dette er en positiv kontrol, der angiver den enzymatiske fordøjelsen var vellykket. Antallet af samlede levedygtige celler og DCs inddrives pr. gram væv er også vist i figur 1B og figur 1 c, henholdsvis. Immunceller repræsenterer mellem 5-30% af de stromale celler før tæthed centrifugering<sup c...

Watch this Scientific Journal Video about Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies at JoVE.com

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

Isolering af dendritiske celler fra det menneskelige kvindelige forplantningsorganerne for Phenotypical og funktionelle studier

Vi beskriver her en metode til at isolere og rense dendritiske celler fra forskellige anatomiske rum i den menneskelige kvindelige forplantningsorganerne for evalueringen af deres phenotypical og funktionelle egenskaber. Denne metode kan tilpasses til at isolere andre immunceller eller dendritiske celler fra andre slimhinden væv.

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

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Isolering af Mouse Lung dendritiske celler

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

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

Isolering og karakterisering af dendritiske celler og makrofager fra mus Tarmen

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

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Flowcytometrisk Isolering af primære murine type II alveolære epitelceller for Funktionelle og molekylære studier

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Isolering af lymfocytter fra Mouse Genital Tract mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

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 ...

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Generering af Umodne, Modne og tolerogeniske dendritiske celler med forskellige Metabolic fænotyper

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

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Generation of Human monocytafledte dendritiske celler fra fuldblod

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

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Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â±

Udvikling og validering af et Ultrasensitive enkelt molekyle Array Digital enzym-forbundet Immunosorbent Assay for menneskelige Interferon-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...