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 och fru Bharti Nehete för makak blod och PBMC prover, Detta arbete har delvis stöd av medel från NIH bidrag AI 46.969, och alla cellodling media producerades av Central Media Laboratory, som finansieras av NIH bidrag CA 16.672.

Förfarandena för detaljerad fenotypisk och funktionella analyser av det totala liksom delar minne av T-celler kan delas in i fyra delar: 1) Cell förberedelse och antigen stimulering, 2) Yta markör färgning, 3) intracellulära cytokiner färgning och 4) Flödescytometri analyser. 1. Cellberedningen och antigena stimulering <ol><li> Både nyligen isolerade samt kryo-bevarade perifera mononukleära blodceller (PBMC) användes i detta protokoll. Den PBMC var isolerade från hepariniserade...

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Lösning och buffertar <ul><li> Ficoll-Hypaque (Histopaquen-1077, Sigma-Aldrich, St Louis, MO) </li><li> RPMI-1640 medium (HyClone laboratorier, Logan, UT) </li><li> Fetalt bovinserum (FBS), (HyClone laboratorier, Logan, UT) </li><li> L-glutamin (Sigma-Aldrich, St Louis, MO) </li><li> Penicillin / streptomycin (Invitrogen, Karlsbad, Kalifornien) </li><li> Trypan blå reagens (Sigma-Aldrich, St Louis, MO) </li><li> 50 mM Tris, PH 8,6, filtreras och lagras vid 4 ° C </li><li> Dulbecco är PBS (DPBS, Ca2/Mg2-free, Life-teknik, Rockville, MD) </li><li> Flöde Tvättbuffert: DPBS komplettera med 2% FBS, förvara vid 4 ° C </li><li> BD Cytofix / Cytoperm Fixering / Permeabilization Solution Kit (BD Biosciences, San Jose, CA) </li></ul> Aktivering agenter <ul><li> Phorbol 12-myristate 13-acetat (PMA) (Sigma-Aldrich, St Louis, MO) </li><li> Ionomycin (Sigma-Aldrich, St Louis, MO) </li><li> Co-stimulerande mAb CD49d, klon 9F10 (BD Biosciences, San Jose, CA) </li><li> Affinitetsrenad antikropp F (ab) 2 fragment av get anti-mus IgG (H + L) (Kierkegaard och Perry Laboratory, Gaithersburg, MD) </li><li> Specifika aktivering agenter kan inkludera peptid pooler, enstaka peptid, eller hela protein </li></ul> Cytokin sekretion hämmare <ul><li> Brefedin A (BFA, Sigma-Aldrich, St Louis, MO) </li><li> Golgi Plug kan användas som alternativ (BD Biosciences, San Jose, CA) </li></ul> Antikroppar <ul><li> Live / döda fastställbara aqua fluorescerande Stain Kit (Invitrogen, Karlsbad, Kalifornien) </li><li> CD3 PE-Cy7 (SP34-2), (BD Biosciences, San Jose, CA) </li><li> CD4-Pacific Blue (OKT4), (eBiosciences (San Diego, Kalifornien) </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

Den rhesus makak-modellen är för närvarande det bästa tillgängliga modellen för hiv-aids vad gäller ökad förståelse för uppkomsten och utvecklingen av vaccin och läkemedel<sup&gt; 1,2,3</sup&gt;. Här beskriver vi en metod för detaljerad fenotypisk och funktionella analyser av cellulära immunsvaret, särskilt intracellulär cytokin produktion av CD4 + och CD8 + T-celler såväl som enskilda delmängder minne. Vi fick exakta kvantitativa och kvalitativa åtgärder för produktion av interferon-gamma (INF-) och interleukin (IL) -2 i både CD4 + och CD8 + T celler från rhesus makak PBMC stimuleras med PMA plus ionomycin (PMA + I). Den cytokin profilerna var olika i de olika undergrupper av minnesceller. Dessutom, under förutsättning att detta protokoll oss känslighet för att påvisa även små fraktioner av antigen CD4 + och CD8 + T celler undergrupper inom PBMC prover från rhesus makaker immuniserade med en hiv-kuvert peptid cocktail vaccin som utvecklats i vårt laboratorium. Den multicolor flödescytometri teknik är ett kraftfullt verktyg för att exakt identifiera olika populationer av T-celler<sup&gt; 4,5</sup&gt; Med cytokin-producerande förmåga<sup&gt; 6</sup&gt; Följande icke-specifik eller antigenspecifika stimulering<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

Vi visar nyttan av multicolor flödescytometri för detaljerad fenotypisk och funktionell karakterisering av den totala såväl som delmängder minne av CD4 + och CD8 + T-celler i rhesus makaker, den ideala modellen för hiv / aids-vaccin studier.

Multicolor flödescytometrisystem Analyser av cellulära immunförsvaret i Rhesus makaker

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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