Name: quantitative high-throughput single-cell cytotoxicity assay for t cells
Uploaded: 2013-02-02T14:00:00
Description: Watch this Scientific Journal Video about Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells at JoVE.com

Forskningen rapporteret i denne publikation blev støttet af National Cancer Institute of National Institutes of Health under Award Number R01CA174385. Indholdet er alene forfatternes ansvar og repræsenterer ikke nødvendigvis de officielle synspunkter National Institutes of Health.

1. Reagenser Forberedelse <ol><li> Forbered RPMI-PLGH ved at blande 500 ml RPMI-1640 og 5 ml hver af penicillin-streptomycin, L-glutamin og HEPES opløsning. </li><li> Forbered R10 opløsning ved at blande RPMI-PLGH med 10% føtalt bovint serum (FBS). The FBS er varmeinaktiveret ved 56 ° C i 30 min før tilsætning. </li><li> Pre-varme ved 37 ° C 50 ml RPMI-PLGH, 15 ml PBS, og 15 ml R10 i sterile koniske rør. </li><li> Fremstilling af arrays af nanowells i PDMS: The silicium master er fremstillet ved hjælp af foto...

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	<li>Louis, C. U., 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=Antitumor+activity+and+long-term+fate+of+chimeric+antigen+receptor-positive+T+cells+in+patients+with+neuroblastoma.">Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma.</a> Blood. 118, 6050-6056 (2011).</li><li>Savoldo, B., 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=CD28+costimulation+improves+expansion+and+persistence+of+chimeric+antigen+receptor-modified+T+cells+in+lymphoma+patients.">CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients.</a> J. Clin. Invest. 121, 1822-1826 (2011).</li><li>Kalos, M., 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=T+cells+with+chimeric+antigen+receptors+have+potent+antitumor+effects+and+can+establish+memory+in+patients+with+advanced+leukemia.">T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.</a> Sci. Transl. Med. 3, 95ra73 (2011).</li><li>Porter, D. L., Levine, B. L., Kalos, M., Bagg, A., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+Antigen+Receptor-Modified+T+Cells+in+Chronic+Lymphoid+Leukemia.">Chimeric Antigen Receptor-Modified T Cells in Chronic Lymphoid Leukemia.</a> N. Engl. J. Med. , (2011).</li><li>Restifo, N. P., Dudley, M. E., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+immunotherapy+for+cancer:+harnessing+the+T+cell+response.">Adoptive immunotherapy for cancer: harnessing the T cell response.</a> Nat. Rev. Immunol. 12, 269-281 (2012).</li><li>Pittet, M. J., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging.">Intravital imaging.</a> Cell. 147, 983-991 (2011).</li><li>Sumen, C., Mempel, T. R., Mazo, I. B., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy:+visualizing+immunity+in+context.">Intravital microscopy: visualizing immunity in context.</a> Immunity. 21, 315-329 (2004).</li><li>Lamoreaux, L., Roederer, M., Koup, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+cytokine+optimization+and+standard+operating+procedure.">Intracellular cytokine optimization and standard operating procedure.</a> Nat. Protoc. 1, 1507-1516 (2006).</li><li>Varadarajan, N., 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=A+high-throughput+single-cell+analysis+of+human+CD8++T+cell+functions+reveals+discordance+for+cytokine+secretion+and+cytolysis.">A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis.</a> J. Clin. Invest. 121, 4322-4331 (2011).</li><li>Ogunniyi, A. O., Story, C. M., Papa, E., Guillen, E., Love, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+individual+hybridomas+by+microengraving+to+discover+monoclonal+antibodies.">Screening individual hybridomas by microengraving to discover monoclonal antibodies.</a> Nat. Protoc. 4, 767-782 (2009).</li><li>Streeck, H., Frahm, N., Walker, B. D. <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+IFN-gamma+Elispot+assay+in+HIV+vaccine+research.">The role of IFN-gamma Elispot assay in HIV vaccine research.</a> Nat. Protoc. 4, 461-469 (2009).</li><li>Varadarajan, N., 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=Rapid,+efficient+functional+characterization+and+recovery+of+HIV-specific+human+CD8++T+cells+using+microengraving.">Rapid, efficient functional characterization and recovery of HIV-specific human CD8+ T cells using microengraving.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 3885-3890 (2012).</li><li>Makedonas, G., Betts, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+in+a+house+of+cards:+re-evaluating+CD8++T-cell+immune+correlates+against+HIV.">Living in a house of cards: re-evaluating CD8+ T-cell immune correlates against HIV.</a> Immunol. Rev. 239, 109-124 (2011).</li><li>Telford, W. G., Komoriya, A., Packard, B. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+analysis+of+apoptosis+by+flow+and+image+cytometry.">Multiparametric analysis of apoptosis by flow and image cytometry.</a> Methods Mol. Biol. 263, 141-160 (2004).</li></ol>

Vi har skitseret protokollen for en high-throughput encellede cytolytisk assay aktiveret via co-inkubation af effektorer og mål i arrays af nanowells (figur 1). Ud over gennemløb en væsentlig fordel ved teknikken er evnen til at overvåge effektor-medieret cytotoksicitet mod ønskede målceller uden behov for målcellen teknik som igen tillader anvendelse af autologe eller matched / primære tumorceller som målceller. Den rumlige indeslutning tillader hentning og efterfølgende kloning af funktionel...

<table border="1"><tbody><tr><td> Navn på reagens / Material </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> RPMI-1640 w / o L-glutamin </td><td> Cellgro </td><td> 15-040-CV </td><td></td></tr><tr><td> Penicillin-streptomycin </td><td> Cellgro </td><td> 30 til 002-CI </td><td> 10.000 IE Penicillin 10.000 ug / ml streptomycin </td></tr><tr><td> L-glutamin </td><td> Cellgro </td><td> 25 til 005-CI </td><td> 200 mM opløsning </td></tr><tr><td> HEPES </td><td> Sigma Aldrich </td><td> H3537 </td><td> 1M </td></tr><tr><td> Føtalt bovint serum (FBS) </td><td> Atlanta Biologicals </td><td> S11150 </td><td> Lot testet </td></tr><tr><td> Cell Tracker Red Stain </td><td> Invitrogen </td><td> C34552 </td><td> 50 ug </td></tr><tr><td> Vybrant DyeCycle Violet Stain </td><td> Invitrogen </td><td> V35003 </td><td> 5 mM </td></tr><tr><td> SYTOX green Nucleic Acid Stain </td><td> Invitrogen </td><td> S7020 </td><td> 5 mM </td></tr><tr><td> Annexin V-Alexa Fluor ® 647 </td><td> Invitrogen </td><td> A23204 </td><td> 500 pi </td></tr><tr><td> Dulbeccos PBS </td><td> Cellgro </td><td> 21-031-CV </td><td> 500 ml </td></tr><tr><td> Noble agar </td><td> DIFCO </td><td> 2M220 </td><td> 100 g </td></tr><tr><td> Trypan Blue </td><td> Sigma Aldrich </td><td> T8154 </td><td> 0,4% væske, sterilfiltreret </td></tr><tr><td> Hæmocytometer </td><td> Hausser Scientifics </td><td> 1492 </td><td> Bright line </td></tr><tr><td> 4-brøndsplade </td><td> Thermo Fisher </td><td> 167.603 </td><td></td></tr><tr><td> Harrick Plasma Cleaner </td><td> Harrick Plasma </td><td> PDC-32G </td><td> Grundlæggende plasma renere </td></tr><tr><td> Observer.Z1 </td><td> ZEISS </td><td&gt; </td><td> Fluorescerende mikroskop (arbejder med de tre dele nedenfor) </td></tr><tr><td> Lambda 10-3 </td><td> Sutter Instrument </td><td></td><td> Filter controller </td></tr><tr><td> Lambda DG-4 </td><td> Sutter Instrument </td><td></td><td> Ultra-High-Speed ​​Wavelength switcher </td></tr><tr><td> Hamamatsu EM-CCD-kamera </td><td> Hamamatsu </td><td> C9100-13 </td><td> CCD-Mikroskop kamera </td></tr><tr><td> 15 ml konisk rør </td><td> BD Falcon </td><td> 352.097 </td><td></td></tr><tr><td> 50 ml konisk rør </td><td> VWR </td><td> 3282-345-300 </td><td></td></tr><tr><td> Nikon Biostation </td><td> Nikon Instruments Inc. </td><td> Biostation IM </td><td></td></tr><tr><td> Glas bunden dyrkningsskål </td><td> Mattek Corporation </td><td> P35G-0 </td><td> 35 mm petriskål, 10 mm mikrobrønd </td></tr></tbody></table>

quantitative high-throughput single-cell cytotoxicity assay for t cells

Kræft immunterapi kan udnytte specificiteten af ​​immunrespons til at målrette og fjerne tumorer. Adoptiv celleterapi (ACT) baseret på den adoptive overførsel af T-celler genmodificerede til at udtrykke et kimært antigen receptor (CAR) har vist betydelig lovende i kliniske forsøg 1-4. Der er flere fordele ved at bruge CAR + T-celler til behandling af cancere, herunder evnen til at målrette ikke-MHC-begrænsede antigener og at funktionalisere T-cellerne for optimal overlevelse, homing og vedholdenhed i værten, og endelig at inducere apoptose af CAR + T-celler i tilfælde af værtsorganismens toksicitet 5. Afgrænse de optimale funktioner CAR + T-celler, der er forbundet med klinisk fordel er afgørende for tilrettelæggelsen af den næste generation af kliniske forsøg. Nylige fremskridt inden for levende dyr billedbehandling som multiphoton mikroskopi har revolutioneret studiet af immun celle funktion in vivo 6,7. Mens disse studier har fremmet vores forståelse af T-cellefunktioner in vivo, T-celle-baserede ACT i kliniske forsøg kræver, at det er nødvendigt at forbinde molekylære og funktionelle egenskaber af T-cellepræparater præ-infusion med klinisk effektivitet efter infusion, ved anvendelse i vitro assays overvågning T-cellefunktioner lignende, cytotoksicitet og cytokinsekretion. Standard flow-cytometri assays er blevet udviklet, at bestemme den overordnede funktion af populationer af T-celler på enkeltcelle-niveau, men disse er ikke egnet til overvågning af konjugatdannelse og levetid eller mulighed for den samme celle at dræbe flere mål 8. Microfabricated arrays designet i biokompatible polymerer, såsom polydimethylsiloxan (PDMS) er en særdeles attraktiv metode til rumligt begrænse effektorer og mål i små volumener 9. I kombination med automatiseret time-lapse fluorescensmikroskopi, thousands af effektor-mål-interaktioner kan overvåges samtidigt af billeddannende individuelle brønde i en nanowell array. Vi præsenterer her en high-throughput metode til overvågning af T-cellemedieret cytotoksicitet på enkeltcelle-niveau, der stort set anvendes til at studere den cytolytiske funktion af T-celler.

Et eksempel på anvendelsen af high-throughput cytolytisk assay er vist i figur 2. Kort fortalt mærket CD19-specifik CAR + T-celler blev co-inkuberet med mærkede muse-EL4-målceller i de enkelte brønde i en nanowell matrix (afsnit 1-5). Et første billede blev optaget på den automatiserede fluorescerende mikroskop for at identificere belægning (effektorer og / eller mål) af hver enkelt nanowell på array (afsnit 6). Billedbehandling blev anvendt til at identificere alle nanowells indeho...

Watch this Scientific Journal Video about Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells at JoVE.com

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Vi beskriver en enkelt-celle high-throughput assay til måling af cytotoksicitet af T-celler, når inkuberet med tumormålceller. Denne fremgangsmåde anvender en tæt, elastomere matrix af sub-nanoliter brønde (~ 100.000 brønde / række) til rumligt begrænse de T-celler og målceller ved definerede forhold og er koblet til fluorescensmikroskopi for at overvåge effektor-mål-konjugation og efterfølgende apoptose.

Kvantitativ Højkapacitetsforskning encellede cytotoksicitetsassay for T-celler

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

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