1. 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 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