Name: multicolor flow cytometry analyses of cellular immune response in rhesus macaques
Uploaded: 2010-04-22T17:35:00
Description: Watch this Scientific Journal Video about Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques at JoVE.com

Dr Pramod Nehete en mevrouw Bharti Nehete voor makaken bloed-en PBMC monsters; Dit werk werd gedeeltelijk ondersteund door de middelen van NIH subsidie ​​AI 46969, en al de celkweek media werden geproduceerd door het Centraal Media Laboratory, dat wordt gefinancierd door NIH subsidie ​​CA 16672.

De procedures voor de gedetailleerde fenotypische en functionele analyse van het totaal en als het geheugen subsets van T-cellen kan worden onderverdeeld in vier delen: 1) Cel voorbereiding en antigene stimulatie, 2) Oppervlakte marker kleuring, 3) Intracellulaire cytokine kleuring en 4) Flowcytometrie analyses. 1. Celpreparaat en antigene stimulatie <ol><li> Zowel vers geïsoleerde als cryo bewaard gebleven perifere mononucleaire bloedcellen (PBMC) werden gebruikt in dit protocol. De PBMC...

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Solution en buffers <ul><li> Ficoll-Hypaque (Histopaquen-1077, Sigma-Aldrich, St. Louis, MO) </li><li> RPMI-1640 medium (Hyclone laboratoria, Logan, UT) </li><li> Foetaal runderserum (FBS), (Hyclone laboratoria, Logan, UT) </li><li> L-glutamine (Sigma-Aldrich, St. Louis, MO) </li><li> Penicilline / streptomycine (Invitrogen, Carlsbad, CA) </li><li> Trypan blauw reagens (Sigma-Aldrich, St. Louis, MO) </li><li> 50 mM Tris, pH 8,6, gefilterd en bewaren bij 4 ° C </li><li> Dulbecco&#39;s PBS (DPBS, Ca2/Mg2-free, Life Technologies, Rockville, MD) </li><li> Flow wasbuffer: DPBS te vullen met 2% FBS, bewaren bij 4 ° C </li><li> BD Cytofix / Cytoperm Fixatie / permeabilisatie Kit Solution (BD Biosciences, San Jose, CA) </li></ul> Activering agenten <ul><li> Forbol 12-myristaat 13-acetaat (PMA) (Sigma-Aldrich, St. Louis, MO) </li><li> Ionomycine (Sigma-Aldrich, St. Louis, MO) </li><li> Costimulatoire mAb CD49d, kloon 9F10 (BD Biosciences, San Jose, CA) </li><li> Affiniteit gezuiverd antilichaam F (ab) 2 fragmenten van geit anti-muis IgG (H + L) (Kierkegaard en Perry Laboratorium, Gaithersburg, MD) </li><li> Specifieke activering agenten kunnen zijn peptide zwembaden, een peptide, eiwit of hele </li></ul> Cytokine secretie-remmers <ul><li> Brefedin A (BFA, Sigma-Aldrich, St. Louis, MO) </li><li> Golgi Plug kan als alternatief worden gebruikt (BD Biosciences, San Jose, CA) </li></ul> Antilichamen <ul><li> Live / dead Muur aqua fluorescerende vlek kit (Invitrogen, Carlsbad, CA) </li><li> CD3 PE-Cy7 (SP34-2), (BD Biosciences, San Jose, CA) </li><li> CD4-Pacific Blue (OKT4), (eBiosciences (San Diego, CA) </li><li> CD8 Alexa700 (RPA-T8), (BD Biosciences, San Jose, CA) </li><li> CD28 PerCP-cy5.5 (L293), (BD Biosciences, San Jose, CA) </li><li> CD95 APC (DX2), (BD Biosciences, San Jose, CA) </li><li> IFN-FITC (B27), (BD Biosciences, San Jose, CA) </li><li> IL-2 PE (MQ1-17H12), (BD Biosciences, San Jose, CA) </li></ul>

multicolor flow cytometry analyses of cellular immune response in rhesus macaques

Het rhesus makaak model is momenteel de best beschikbare model voor HIV-AIDS met betrekking tot het begrijpen van de pathogenese, alsmede voor de ontwikkeling van vaccins en therapeutica<sup&gt; 1,2,3</sup&gt;. Hier beschrijven we een methode voor de gedetailleerde fenotypische en functionele analyse van de cellulaire immuunrespons, in het bijzonder intracellulaire cytokine productie door CD4 + en CD8 + T-cellen, alsook de individuele geheugen subsets. We nauwkeurige kwantitatieve en kwalitatieve maatregelen voor de productie van interferon-gamma (INF-) en interleukine (IL) -2 verkregen in zowel de CD4 + en CD8 + T-cellen uit het rhesus makaak PBMC gestimuleerd met PMA plus ionomycine (PMA + I). De cytokine profielen zijn verschillend in de verschillende subsets van geheugencellen. Bovendien is dit protocol heeft ons de gevoeligheid om zelfs kleine fracties van antigeen specifieke CD4 tonen + en CD8 + T-cel subsets binnen de PBMC monsters van resusmakaken geïmmuniseerd met een HIV-envelop peptide cocktail-vaccin ontwikkeld in ons laboratorium. De multicolor flowcytometrie techniek is een krachtig hulpmiddel om nauwkeurig te identificeren verschillende populaties van T-cellen<sup&gt; 4,5</sup&gt; Met cytokine-producerende vermogen<sup&gt; 6</sup&gt; Volgende niet-specifieke of antigeen-specifieke stimulatie<sup&gt; 5,7</sup&gt;.

Watch this Scientific Journal Video about Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques at JoVE.com

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

Tonen we het nut van multicolor flowcytometrie voor gedetailleerde fenotypische en functionele karakterisering van het totaal en als het geheugen subsets van CD4 + en CD8 + T-cellen in rhesus makaken, het ideale model voor HIV / AIDS-vaccin studies.

Multicolor Flow Cytometry Analyses van de cellulaire immuunreactie in resusmakaken

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

Research

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