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

Este trabalho foi financiado pela subvenção NIH R01AI118694 para Michael R. Betts, que inclui o prêmio sub 566950 para Yuri Sykulev. Agradecemos a Sidney Kimmel Cancer Center Bioimaging recurso compartilhado pelo excelente apoio.

Este estudo foi realizado em conformidade com a declaração de Helsinque. Foi obtido consentimento escrito de todos os participantes, e amostras de sangue e linfonodo foram adquiridas com a aprovação do Conselho de revisão institucional da Universidade da Pensilvânia (IRB #809316, IRB # 815056). Todos os seres humanos eram adultos. Amostras de sangue de cordão foram gentilmente cedidas pelo trabalho e entrega do departamento de obstetrícia & ginecologia na Universidade de Thomas Jefferson. Todas as amostras foram ...

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A nova técnica descrita aqui utiliza reagentes semelhantes necessários para construir bilayers planares no fluxo convencional célula5 e pode ser aplicada com sucesso para executar a imagem da principal célula T humana – BICAMADA interfaces3,4 ,15. A técnica oferece uma redução significativa no uso do moléculas fluorescentes e necessita de 10 – 20 x menos células T em comparação com um flux...

Conhecimento científico das características estruturais de sinapses imunes de células T e sua ligação com a atividade funcional das células T foi gerado principalmente a partir do estudo de linhagens celulares e clones derivado do PB. A que ponto esses achados se relacionam com primárias células T obtidos de sangue ou tecidos linfoides humanos permanece obscuro, como as interfaces sinápticas de células T que residem nos tecidos linfoides e outros não foram analisadas até agora. Importante, dados emergentes sugerem que o residente em tecidos e órgãos linfoides-derivado de células T podem ter diferenças significativas em seu fenótipo e atividade funcional em comparação com aqueles em PB6,7. Isto solidificou ainda mais a necessidade de compreender melhor as características da interface sináptica de células T na primárias células T humanas.
Para este fim, nós desenvolvemos uma abordagem de escala mini novela explorar bilayers do lipid construídos em slides de fluxo multicanal, permitindo-na realizar a imagem de interfaces de T-celular/BICAMADA com menos de 105 primário T células isoladas de humano PB e LN. Esta nova técnica permite o estudo das propriedades biofísicas de primário T-célula sinápticas interfaces humanas a fim de melhor modelo e perceber na vivo interações célula-célula.

<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

A atual compreensão da dinâmica e características estruturais das interfaces sinápticas de células T tem sido largamente determinadas com o uso de vidro apoiados bilayers planares e in vitro-T células derivadas de clones ou linhas1,2 ,3,4. Como estes resultados aplicam-se para as células humanas primárias T isoladas do sangue ou linfoides tecidos não é conhecido, em parte devido a dificuldades na obtenção de um número suficiente de células para análise5. Aqui abordamos isto através do desenvolvimento de uma técnica de slides de fluxo multicanal para construir bilayers do lipid planar contendo moléculas de aderência e ativando a explorar. A baixa altura dos slides fluxo promove a sedimentação celular rápida para sincronizar o acessório de celular: BICAMADA, permitindo aos pesquisadores estudar a dinâmica da formação sináptica interface e a cinética da liberação de grânulos. Podemos aplicar esta abordagem para analisar a interface sináptica de sómente 104 105 primária criopreservado T células isoladas de linfonodos (LN) e sangue periférico (PB). Os resultados revelam que a técnica de bicamada lipídica planar romance permite o estudo das propriedades biofísicas de primárias células T humanas, derivadas do sangue e tecidos no contexto da saúde e na doença.

Primeiro, comparamos a estrutura da interface sináptica formada por ativadas CD8 cabo-hemoderivados+ T células expostas a bilayers do lipid construíram ou fluxo em grande escala tradicional em sistemas de células (veja a Tabela de materiais para obter detalhes)1 ,2,3,4 ou em slides de fluxo multicanal. Os bilayers continham fluor...

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

Avaliação da Interface sináptica de células T humano primário do sangue periférico e tecido linfoide

O protocolo descreve uma técnica para estudar a capacidade das células T policlonais humanas de primário para formar sinápticas interfaces usando bilayers do lipid planar. Usamos esta técnica para mostrar a capacidade de formação de sinapse diferencial de células T humanas primárias derivado dos gânglios linfáticos e sangue periférico.

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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