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OMIP-005

Quality and phenotype of antigen-responsive rhesus macaque T cells

Kathryn E. Foulds, Mitzi Donaldson, Mario Roederer Cytometry PART A, Volume 81A, Issue 5, 360-361 (2012)

PURPOSE: The primary eight-color panel was designed to measure IFN╬│, IL-2, and TNF production from viable CD4 and CD8 T cells from rhesus macaques in preclinical vaccine studies. An 11-color variant also allows for the assessment of memory subsets based on surface expression of CD28, CD45RA, and CCR7. The panel was optimized not only for use on cryopreserved peripheral blood mononuclear cell (PBMC) samples but also works well on fresh PBMC samples, cryopreserved tissue samples, and fresh tissue samples that have been treated with RBC lysis buffer (Table 1). The eight-color panel and associated staining procedure were tested in a formal qualification study and shown to be highly reproducible with low interaliquot, interday, and interanalyst variability according to the qualification criteria (manuscript in preparation).

CELL TYPE: Peripheral Blood Mononuclear Cells

MACHINE: BD LSR2

Marker Target Clone Fluorochrome Purpose
Dead cells Rhesus macaque Live/Dead Fix Aqua viability
CD4 Rhesus macaque S3.5 Qdot 605 T cell
CD45RA Rhesus macaque L48 PE-Cy7 memory
CD28 Rhesus macaque 28.2 PE-Cy5 memory
CD8 Rhesus macaque RPA-T8 Pacific Blue T cell
CD3 Rhesus macaque SP34-2 APC-Cy7 T cell
CD69 Rhesus macaque TP1.55.3 ECD background reduction
IFN-gamma Rhesus macaque B27 APC cytokines
IL-2 Rhesus macaque MQ1-17H12 PE cytokines
TNF alpha Rhesus macaque Mab11 FITC cytokines
ccr7 Rhesus macaque 150503 Alexa Fluor 680 memory