Fitc conjugated anti ccr7 clone 150503

Cell surface stains were performed by washing cells twice with FACS buffer, 1% FBS in 1× PBS, followed by incubation with human Cohn's fraction IgG (Sigma-Aldrich, G4386) for 10 minutes to reduce non-specific binding. Samples were then washed with FACS buffer twice before incubation in the appropriate mixture of antibodies for 30 min at 4°C. After incubation, the cells were washed three times and resuspended in FACS buffer before samples were acquired on a C6 Accuri flow cytometer (BD Biosciences, Ann Arbor, MI). Expression of MICA/B on cell lines was quantified using PE-conjugated anti-MICA/MICB antibody clone 6D4 (Biolegend, 320906). T cell subsets were identified using anti-CD3 clone OKT3 conjugated with PerCP 710 (eBiosciences, 46-0037-42), PE conjugated anti-CD45RO clone UCHL1 (Biolegend, 304206), and FITC-conjugated anti-CCR7 clone 150503 (R&D Systems, FAB197F). When intracellular staining was performed, cell surface stains were completed before cells were fixed and permeabilized using a Fix/Perm staining kit (eBioscience, 00-5523-00) followed by intracellular staining for IFNγ with APC anti-human IFNγ clone B27, (Biolegend, 506510) or isotype control APC msIgG1 clone MOPC-21 (Biolegend, 400120) per the manufacturer's instructions. All data acquisition and analysis was done using the Accuri flow cytometry software (BD Biosciences, Ann Arbor, MI).