Name: isolation and flow cytometric analysis of human endocervical gamma delta t cells
Uploaded: 2017-02-06T10:30:00
Description: Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

Vi takker Miami WIHS deltagerne for deres vilje til å delta i dette arbeidet. Dataene i dette manuskriptet ble samlet av Miami kvinner Interagency HIV Study (WIHS). Innholdet i denne publikasjonen er utelukkende ansvaret til forfatterne og representerer ikke de offisielle visningene av National Institutes of Health (NIH). Dette arbeidet ble støttet av National Institute of Allergy og smittsomme sykdommer og kvinner Interagency HIV infeksjon Study (WIHS) [tilskuddet antallet U01 AI103397], National Institute of Allergy og smittsomme sykdommer, National Institutes of Health [bevilgning antall P30AI073961], Nasjonalt Senter for Advancing translasjonsforskning Sciences og National Institute on minoritetshelse og helseforskjeller, National Institute of Health [bevilgning antall UL1TR000460 og 1KL2TR000461], og National Institute of Child Health and human Development, National Institute of Health [bevilgning antall K23HD074489].

Studieaktiviteter fant sted ved University of Miami HIV Research Unit i samarbeid med Miami kvinner HIV Interagency Study (WIHS) og Miami Senter for AIDS forskning (CFAR) .Institutional Review Board (University of Miami Miller School of Medicine) godkjenning ble innhentet før til rekruttering og noen vurdering eller studierelaterte prosedyrer. 1. endocervikale Brush Prøvetaking MERK: Deltakerne gjennomgikk en vaginal undersøkelse utført av utdannet MD eller gyn...

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Vurdering av kvinnelig lavere kjønnsorgan mucosal sårbarhet er en viktig del av kliniske studier som omhandler risiko for HIV oppkjøp og overføring. Vanligvis FGT sårbarhet for HIV-infeksjon blir evaluert ved å måle løselig immunmodulatorer i genital sekreter (cytokiner, kjemokiner og antimikrobielle peptider) 12, 13. Men disse biomarkører er bare en indikasjon på vaginal betennelse, påvirkes av fysiologiske og patologiske hendelser, og ikke alltid re...

Vi har utviklet metodikk for å bruke endocervikale penselprøver for å vurdere intraepitelial GD T-celler. Den endocervikale kanalen er omgitt av et enkelt lag av søyle epitel. Intraepitelial lymfocytter representerer front lymfocytter bosatt i epiteliale lag som raskt kan initiere immunresponser ved å støte patogene mikrober 7, 8. Forstå immunologiske hendelsene i endocervikale intraepitelial rommet er viktig for utformingen av effektive strategier for å forebygge infeksjoner, inkludert HIV. Vår metode kan brukes for fersk innsamlede humane endocervikale prøver samt frosne endocervikale celler for å ytterligere undersøke hvilken rolle intraepitelial GD T-celler hos kvinner med HIV-infeksjon eller med risiko for HIV-smitte. Hovedmålet med denne protokollen er å oppdage nye immunologiske hendelser i endocervikale intraepitelial rommet og til evaluate GD T-celle responser som en markør for slimhinne sårbarhet hos kvinner med HIV-infeksjon. De betydelige kunnskapshull rundt FGT immunitet er delvis på grunn av vanskeligheter med å lykkes innsamling og behandling av slimhinneprøver. Videre er den komplekse heterogeniteten av mukosale immunceller, og deres sammenhengene med hverandre, er store utfordringer for identifisering av klinisk relevante målinger som reflekterer tilstanden og evnen av immunsystemet. Vi utviklet en metode for å skaffe høy-rene intraepitelial GD T-celler som kan bli ytterligere analysert ved hjelp av svært multipleksede, encellede teknologier som kan være avgjørende for å identifisere de underliggende mekanismene for FRT immunitet.

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			<td height="42" style="height:42px;width:216px;">Cytobrush Plus GT&#160;</td>
			<td style="width:147px;">(CareFusion, San Diego, CA, USA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">IMDM,Iscove&#39;s Modified Dulbecco&#39;s Medium&#160;</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">12440061</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Beckman Coulter automated cell counter (The Vi-CELL Series Cell Viability Analyzer</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">496178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO, Dimethyl sulfoxide&#160;</td>
			<td style="width:147px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS, Fetal Bovine Serum</td>
			<td style="width:147px;">Invitrogen, Gibco</td>
			<td style="width:119px;">26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:147px;">Invitrogen, Gibco</td>
			<td align="right" style="width:119px;">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">A-9647</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LIVE/DEAD Fixable Yellow Dead Cell Stain kit&#160;</td>
			<td style="width:147px;">Life Technologies,</td>
			<td style="width:119px;">L349S9</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1% paraformaldehyde (Sigma-Aldrich</td>
			<td style="width:147px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">D6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fortessa flow cytometer&#160;</td>
			<td style="width:147px;">Becton Dickinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraComp eBeads</td>
			<td style="width:147px;">eBioscience</td>
			<td style="width:119px;">01-2222-42</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">ArC Amine Reactive Compensation Beads (Life Technologies, Grand Island, NY, USA)</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:119px;">A10346</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-TCR gamma/delta MicroBead Kit&#160;</td>
			<td style="width:147px;">Milteny Miltenyi Biotec Inc.</td>
			<td style="width:119px;">130-050-701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MS column</td>
			<td style="width:147px;"></td>
			<td style="width:119px;">103-042-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS separator</td>
			<td style="width:147px;"></td>
			<td align="right" style="width:119px;">4124</td>
			<td></td>
		</tr>
	</tbody>
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isolation and flow cytometric analysis of human endocervical gamma delta t cells

Den kvinnelige skjede (FRT) mucosal immunsystemet fungerer som første forsvarslinje. Bedre kunnskap om genital slimhinne er derfor viktig for å forstå patogenitet av ulike patogener inkludert HIV. Gamma Delta (GD) T-celler er prototypen på &#39;ukonvensjonelle&#39; T-celler og representerer en relativt liten del av T-celler som er definert av deres uttrykk for heterodimere T-celle reseptorer (TCR) består av gamma og delta kjeder. Dette skiller dem fra de klassiske og mye bedre kjent CD4 + T-hjelpeceller og CD8 + cytotoksiske T-celler som er definert av alfa-beta TCR. GD-T-celler viser ofte vev-spesifikk lokalisering og anrikes i epitel. GD T-celler orkestrere immunresponser i betennelse, svulst overvåking, smittsomme sykdommer, og autoimmunitet. Her presenterer vi en metode for å reproduserbart isolere og analysere menneskelige endocervikale intraepitelial GD T-lymfocytter. Vi hahar brukt endocervikale cytobrush prøver fra kvinner som deltar i kvinnenes Interagency HIV infeksjon Study (WIHS). Kunnskap om GD T-celler interaksjoner under forhold der det er en fornærmelse mot vaginal slimhinnene kan brukes til klinisk studie hvor mucosal sårbarhet er adressert, herunder utvikling av vaginal microbicides.In tillegg har kunnskap om slimhinnene GD T-celle responser potensialet for anvendelse av GD T-celle-basert immunterapi ved behandling av infeksjonssykdommer.

Nylig, analyserte vi endocervikale intraepitelial GD T-celler, og vi var de første til å rapportere om tap av endocervikale gamma delta T-celler hos HIV-smittede kvinner 9, 11. Her beskriver vi protokollen for å isolere og analysere undergruppe av endocervikale gamma delta T-celler. Som vist i figur 1, kan GD T-celler lett bli funnet i humane endocervikale celleprøver. <p class="jove_content" fo:keep-toge...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Isolasjon og Flowcytometriske analyse av endocervikale Gamma Delta T-celler

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

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