Name: assessment of the synaptic interface of primary human t cells from peripheral blood and lymphoid tissue
Uploaded: 2018-07-30T15:00:00
Description: Watch this Scientific Journal Video about Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue at JoVE.com

Dit werk werd gesteund door de R01AI118694 NIH-subsidie aan Michael R. Betts, waaronder sub award 566950 naar Yuri Sykulev. Wij danken de Sidney Kimmel Cancer Center Bioimaging gedeelde Resource voor hun uitstekende steun.

Deze studie werd uitgevoerd overeenkomstig de verklaring van Helsinki. Schriftelijke toestemming is verkregen van alle deelnemers, en bloed-en lymfeklier werden verworven met de goedkeuring van de institutionele Review Board aan de Universiteit van Pennsylvania (IRB #809316, IRB # 815056). Alle proefpersonen waren volwassenen. Koord bloedmonsters werden beschikbaar gesteld door arbeid en levering van de afdeling Obstetrie & gynaecologie aan de Thomas Jefferson University. Alle monsters waren-geïdentificeerde.
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De nieuwe techniek hier beschreven maakt gebruik van soortgelijke reagentia moeten bouwen vlakke bilayers in de conventionele stroom cel5 en met succes kan worden toegepast voor het uitvoeren van de beeldvorming van primaire menselijke T-cel-dubbelgelaagde interfaces3,4 ,15. De techniek biedt een significante vermindering van het gebruik van fluorescerend moleculen en vereist 10 – 20 x minder T-cellen ...

Wetenschappelijke kennis van de structurele kenmerken van T-cel immuun synapsen en hun link naar de functionele activiteit van T cellen is gegenereerd voornamelijk uit de studie van cellijnen en klonen afgeleid van PB. In welke mate deze bevindingen hebben betrekking op primaire T-cellen verkregen uit blijft bloed of menselijke lymfoïde weefsels onduidelijk, zoals de synaptische interfaces van T-cellen die woonachtig zijn in de lymfoïde en andere weefsels hebben tot nu toe niet is geanalyseerd. Belangrijker, suggereren opkomende gegevens dat weefsel-ingezetene en lymfoïde-orgel-afgeleide T-cellen kunnen aanzienlijke verschillen in hun fenotype en functionele activiteit in vergelijking met PB6,7. Dit heeft verder verhard de noodzaak om de functies van de T-cel synaptic interface in primaire menselijke T cellen beter te begrijpen.
Daartoe hebben we een aanpak van de nieuwe mini schaal benutten van lipide bilayers ingebouwd in meerkanaals stroom dia's, zodat we kunnen uitvoeren van de beeldvorming van T-cel/dubbelgelaagde interfaces met minder dan 105 primaire T cellen geïsoleerd van menselijke PB en LN. Deze nieuwe techniek maakt het mogelijk de studie van de biofysische eigenschappen van primaire menselijke T-cel synaptic interfaces om beter model en in vivo cel interacties begrijpen.

<table>
	<tbody>
		<tr>
			<td>CD4 T cell isolation kit, human</td>
			<td>Miltenyl Biotec</td>
			<td>130-096-533</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8 T cells Isolation Kit, human</td>
			<td>Miltenyl Biotec</td>
			<td>130-096-495</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPC</td>
			<td>Avanti Polar Lipids</td>
			<td>850375C</td>
			<td></td>
		</tr>
		<tr>
			<td>DOGS NTA</td>
			<td>Avanti Polar Lipids</td>
			<td>790528C</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinyl Cap PE</td>
			<td>Avanti Polar Lipids</td>
			<td>870273C</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum Albumin</td>
			<td>Octapharma USA</td>
			<td>NDC 68982-643-01</td>
			<td></td>
		</tr>
		<tr>
			<td>sticky-Slide VI 0.4</td>
			<td>ibidi</td>
			<td>80608</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips for sticky-Slides</td>
			<td>ibidi</td>
			<td>10812</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioptech FCS2 Chamber</td>
			<td>Bioptech</td>
			<td>060319-2-03</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD3 antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>16-0037-81</td>
			<td>OKT3 clone, hybridoma cells are available from ATCC</td>
		</tr>
		<tr>
			<td>anti- CD28 antibody</td>
			<td>Genetex</td>
			<td>GTX14664</td>
			<td>9.3 clone</td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma</td>
			<td>C5890</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-PEO4-NHS</td>
			<td>Thermo Fisher Scientific</td>
			<td>21329</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 protein labeling kit with column for labeled protein purification</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10235</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 protein labeling kit with column for labeled protein purification</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10238</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Cy5 NHS Ester</td>
			<td>GE Life Science</td>
			<td>PA15101</td>
			<td></td>
		</tr>
		<tr>
			<td>pMT/V5-His A, B, C Drosophila Expression Vectors</td>
			<td>Thermo Fisher Scientific</td>
			<td>V412020</td>
			<td></td>
		</tr>
		<tr>
			<td>pcopneo, G418 Drosophila expression vector for positive selection</td>
			<td>ATCC</td>
			<td>37409</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum free Drosophial media Insect-XPRESS</td>
			<td>Lonza</td>
			<td>12-730Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridoma YN1/1.7.4</td>
			<td>ATCC</td>
			<td>CRL1878</td>
			<td>The hybridoma secrets antibody against ICAM-1.</td>
		</tr>
		<tr>
			<td>Cyanogen bromide-activated-Sepharose 4B</td>
			<td>Sigma-Aldrich</td>
			<td>C9142</td>
			<td>Utilized for preparation of Sepharose with covelently bound anti-ICAM antibody.</td>
		</tr>
		<tr>
			<td>MasterFlex tangential flow concentrator</td>
			<td>Cole-Parmer</td>
			<td>77601-60 7592-40</td>
			<td>Used for ICAM-1 containing supernatant concentration and dialysis of ICAM-1 containing supernant</td>
		</tr>
		<tr>
			<td>Centramate Lab Tangential Flow Systems</td>
			<td>Pall Laboratory</td>
			<td>FS002K10 OS010T12 FS005K10</td>
			<td>Used for ICAM-1 containing supernatant concentration and dialysis of ICAM-1 containing supernant</td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing</td>
			<td>Spectra/Por</td>
			<td>131384</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain from papaya latex</td>
			<td>Sigma</td>
			<td>P3125</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-human antibody against CD107a</td>
			<td>BD Bioscences</td>
			<td>555798</td>
			<td>Clone H4A3</td>
		</tr>
		<tr>
			<td>Ansell Natural Blue Gloves</td>
			<td>Fisher Scientific</td>
			<td>19-014-539</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Polypropylene Scissor-Type Forceps</td>
			<td>Thermo Fisher Scientific</td>
			<td>6320-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>ProZyme</td>
			<td>SA10</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Nikon TiE inverted microscope equipped with PFS for long-term image stability control, 60x oil objectives, 4 lasers with excitation lines at 405, 458, 488, 514, 561, and 640 nm, 2 GaAsP detectors and 2 high sensitivity PMTs, DIC transmitted light, Programmable X,Y,Z stage for multiple positions and stitching of large areas, time lapse functions, Tokai-Hit temperature and CO2-controlled chamber for live imaging, and anti-vibration isolation table</td>
		</tr>
		<tr>
			<td>TIRF microscope</td>
			<td>Andor</td>
			<td></td>
			<td>Andor Revolution XD system equipped with Nikon TIRF-E illuminator, Lasers with 405,488,561 and 640 lines, DIC transmitted light, Yokogawa CSU-X1 spinning disk head for confocal imaging, 100/1.49 NA objective, Andor iXon X3 EM-CCD camera, objective heater, and a piezoelectric motorized stage with Perfect Focus System (PFS)</td>
		</tr>
		<tr>
			<td>MetaMorph Premier Image Analysis Software</td>
			<td>Molecular devices</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of the synaptic interface of primary human t cells from peripheral blood and lymphoid tissue

Het huidige begrip van de dynamiek en structurele kenmerken van T-cel synaptic interfaces is grotendeels bepaald door het gebruik van glas-gesteund vlakke bilayers en in vitro-afgeleide T-cell klonen of lijnen van1,2 ,3,4. Hoe deze bevindingen van toepassing zijn op de primaire menselijke T-cellen geïsoleerd van bloed of lymfoïde weefsels niet bekend is, deels te wijten aan de grote moeilijkheden bij het verkrijgen van een voldoende aantal cellen voor analyse5. Hier aanpakken we dit door middel van de ontwikkeling van een techniek die exploitatie van meerkanaals stroom dia's om te bouwen van vlakke lipide bilayers met activeren en hechting van de moleculen. De geringe hoogte van de stroom dia's bevordert snelle cel sedimentatie om te synchroniseren van cel: dubbelgelaagde bijlage, waardoor onderzoekers de dynamiek van de formatie van het synaptische interface en de kinetiek van de release van de korrels te gaan studeren. We passen deze benadering voor het analyseren van de synaptische interface van zo weinig als 104 naar 105 primaire cryopreserved T cellen geïsoleerd uit lymfklieren (LN) en perifeer bloed (PB). De resultaten onthullen dat de roman vlakke lipide dubbelgelaagde techniek de studie van de biofysische eigenschappen van primaire menselijke T-cellen afgeleid van bloed en de weefsels in het kader van gezondheid en ziekte maakt.

Eerst, vergeleken we de structuur van de synaptische interface gevormd door geactiveerde koord-bloed-afgeleide CD8+ T cellen blootgesteld aan lipide bilayers gebouwd ofwel in traditionele grootschalige stroom cel systemen (Zie de Tabel van materialen voor details)1 ,2,3,4 of in meerkanaals stroom dia's. De bilayers bevatte TL-geëtik...

Watch this Scientific Journal Video about Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue at JoVE.com

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

Beoordeling van de synaptische Interface van primaire menselijke T-cellen uit perifeer bloed en lymfoïde weefsel

Het protocol beschrijft een techniek om te bestuderen van de mogelijkheid van primaire polyklonale menselijke T cellen vormen van synaptic interfaces met behulp van vlakke lipide bilayers. We gebruiken deze techniek te laten zien van de differentiële synapse formatie vermogen van menselijke primaire T-cellen afgeleid van lymfeklieren en perifeer bloed.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

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