A. B. EXP #1: 3 donors EXP #2: 3 donors EXP #3: 6 donors EXP #4:

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A. B. EXP #1: 3 donors EXP #2: 3 donors EXP #3: 6 donors EXP #4: SAMPLE TYPE: activated, fixed, and frozen whole blood heparinized whole blood cryopreserved PBMC SAMPLE TYPE: cryopreserved PBMC ACTIVATION ANTIGENS: CMV pp65 peptide mix SEB no antigen STAINS: IFNg FITC/CD69 PE/ CD4 PerCP-Cy5.5/CD3 APC CD8 PerCP-Cy5.5/CD3 APC ACTIVATION ANTIGENS: CMV pp65 peptide mix CEF peptide mix no antigen STAINS: IFNg FITC/CD69 PE/ CD4 PerCP-Cy5.5/CD3 APC CD8 PerCP-Cy5.5/CD3 APC CD4 FITC/IFNg+IL-2 PE/ (shipped on liquid nitrogen) (shipped room temp. to U.S. sites only) (shipped on liquid nitrogen) Plate with lyophilized Ag+BFA 96-well plate activation and staining (shallow well for PBMC, deep well for whole blood) 6 h @ 37oC, EDTA, FACS Lyse, FACS Perm 2 Plate with lyophilized Ab Flow cytometry Flow cytometry ANALYSIS: Manual analysis by each site Centralized automated analysis ANALYSIS: Manual analysis by each site Centralized automated analysis