Anti cd107 pe

Thawed PBMC were cultured overnight in RPMI1640 with 10% FBS (Serum Source International), 1% penicillin/streptomycin, 2 mM L-glutamine and 10 mM HEPES (Mediatech). The next day, PBMCs were counted and cultured in the presence of anti-CD107-PE (BD Biosciences) with or without K562 cells (ATCC, Manassas, VA) as described ^{7 (link)} without addition of cytokines, then stained with EMA and lineage-specific antibodies as described above.

Thawed PBMCs were cultured overnight in RPMI 1640 with 10% FBS (Serum Source International), 1% penicillin/streptomycin, 2 mM L-glutamine and 10 mM HEPES (Mediatech). The next day, PBMCs were counted and cultured in the presence of anti-CD107-PE (BD Biosciences) with or without target K562 cells (ATCC, Manassas, VA) as described^{17 (link)} without addition of cytokines, then stained with EMA and lineage-specific antibodies as described above.

Peripheral blood mononuclear cells were co-cultured at a 1:5 ratio with un-pulsed autologous dendritic cells, or E75 peptide-pulsed dendritic cells, or E90 peptide-pulsed dendritic cells in AIMV 10% human AB serum and unlabeled anti-CD28/49 (BD Biosciences, San Jose CA) and anti-CD107-PE (Pharmingen, San Jose CA) for an hour. Brefeldin A and GolgiStop (BD Biosciences) 5 mg/ml each were added and the staining and stimulation were continued for an additional 5 hrs. Following FACS lyse and FACS (Sigma) permeabilization steps, the PBMC:DC stimulated cells were blocked with 200 ng/ml murine IgG before staining with anti-CD8 PerCP and anti-IFN-γ FITC (BD Biosciences) and fixing in 1% formalin (Polysciences, Warrington PA) in PBS before acquisition on FACSCaliber^™ flow cytometer (BD Biosciences).