< OMIPS Directory
OMIP-016

Characterization of antigen-responsive macaque and human T-cells

Sabrina Guenounou, Nathalie Bosquet, Claudia J. Dembek, Roger Le Grand, Antonio Cosma Cytometry PART A, Volume 83A, Issue 2, 182-184 (2013)

PURPOSE: The present panel was optimized to assess the quality and phenotype of antigen-specific CD4 and CD8 T cells in both cynomolgus macaques and humans. The use of an identical protocol for specimens collected in the two species allows for an immediate translation of research from the macaque model to humans. Our protocol works well with cryopreserved and freshly collected PBMC. Following the fixing and permeabilization procedure, we introduced a freezing step that breaks the experimental procedure and allows the shipment of freshly stimulated and permeabilized samples to facilities equipped with instruments able to measure ten distinct fluorescences. Our procedure is thus adapted to multicenter studies where stimulation is performed on fresh PBMC and flow cytometry acquisition is done in a centralized facility.

CELL TYPE: Fresh or cryopreserved PBMCs

MACHINE: BD LSR2

Marker Target Clone Fluorochrome Purpose
Dead cells Cynomolgus macaque Live/Dead Fix Blue viability
CD3 Cynomolgus macaque SP34-2 APC-Cy7 T cell subset
CD4 Cynomolgus macaque L200 PerCP-Cy5.5 T cell subset
CD8 Cynomolgus macaque RPA-T8 V500 T cell subset
CD45RA Cynomolgus macaque L48 PE-Cy7 Differentiation
CD40L Cynomolgus macaque TRAP1 FITC Cytokines/Chemokine/Activation marker
MIP-1 beta Cynomolgus macaque D21-1351 PE Cytokines/Chemokine/Activation marker
IFN-gamma Cynomolgus macaque B27 V450 Cytokines/Chemokine/Activation marker
TNF alpha Cynomolgus macaque Mab11 Alexa Fluor 700 Cytokines/Chemokine/Activation marker
IL-2 Cynomolgus macaque MQ1-17H12 APC Cytokines/Chemokine/Activation marker