After whole blood was treated in the conditions as described under Human Blood Sample Preparation for 16 hours, the blood samples were transferred to 50 mL tubes with Hypaque Ficoll (Invitrogen, Carlsbad, CA), and then centrifuged at 1500 x g for 30 min. The buffy coat interface was carefully removed by pipetting and the residue washed with DPBS twice. Peripheral blood mononuclear cells (PBMCs) were then manually counted by microscopy and viability checked with Trypan Blue staining. Culture medium consisted of RPMI 1640 (Sigma Aldrich, St Louis, MO) which was supplemented with 10% fetal bovine serum, L-glutamine, and antibiotic/antimycotic agents (Thermo Fisher Scientific, Waltham, MA) at a concentration of 5 x 106 cells/mL. PBMC’s were cultured in a 24 well plate (Costar, Corning, NY). Cells were then stimulated with a pre-mixed cocktail of Phorbol myristate acetate (PMA), Ionomycin and Brefeldin A (Leukocyte Activation Cocktail with BD Golgiplug™ BD Biosciences, La Jolla, CA) for 6 hours. After stimulation, cells were incubated with either FITC-conjugated CD4 or CD8 antibodies (BD Biosciences, La Jolla, CA) for 20 min and fixed with 300 μL of 1% formalin at 4°C. Following fixation, cells were permeabilized with 10% methanol for 1 h, stained for PE-conjugated IFN-γ antibodies or the appropriate IgG1 isotype control (eBioscience, San Diego, CA) for 30 min and then analyzed by flow cytometry as described above.