For the CTL assay, frozen PBMC samples from each time point were thawed and cultured for 24 h prior to use in complete medium consisting of RPMI 1640 (Invitrogen) supplemented with

nonessential amino acids (Invitrogen), penicillin/streptomycin (Invitrogen) and 10% heat-inactivated fetal bovine serum (FBS; Invitrogen) at 37 °C, in a 5% CO2 incubator. Autologous tumor cells were maintained in a 6 well plate coated with matrigel matrix (BD bioscience, San Jose, CA) in DMEM (Invitrogen) supplemented with penicillin/streptomycin and 10% FBS. The day of the assay, effector cells were incubated with target cells in complete RPMI 1640 media in 12 × 75 mm Facs tubes (BD bioscience) see more for 5 h at 37 °C in 5% CO2. The effector/target cell ratio used was 10:1 with 1 × 105 PBMCs. Effector cells from each time point were cultured alone (no targets) as control for spontaneous degranulation and IFNγ elaboration. A representative background degranulation response is shown in Fig. 2B, left panel. FITC conjugated anti-CD107b

antibody (AbD Serotec, Oxford, UK) or IgG1 isotype control (AbD Serotec) was added at the beginning of the co-culture. After a 1-h coincubation, Monensin (1:100 dilution; BD Biosciences) and Brefeldin A (3 μg/ml final concentration; eBioscience) were added C646 in vivo for the last 4 h of incubation [26]. Following incubation, cell suspensions were washed with ice-cold PBS and stained for with anti-CD8 check antibody conjugated to Alexa Fluor 700 (AbD Serotec) for 30 min at 4 °C. Samples were then fixed and permeabilized using BD Cytofix/Cytoperm kit (BD bioscience) and stained for intracellular IFNγ with the cross-reactive anti-bovine IFNγ antibody conjugated to PE (AbD Serotec). For detection of Tregs, frozen PBMCs were thawed and added at 1 × 105 in 12 × 75 mm Facs tubes. Cell surface staining was done using Pacific Blue-conjugated anti-dog CD4 antibody (AbD Serotec) or IgG1 isotype control (AbD Serotec) at 4 °C for 30 min. Following incubation, cell suspensions were

washed with cold PBS and resuspended in fixation permeabilization working solution (Foxp3 staining buffer set, eBioscience) overnight. The next day cells were washed with permeabilization buffer (Foxp3 staining buffer set, eBiosceince) followed by intracellular staining with a cross-reactive anti-mouse Foxp3 PeCy-5 conjugated antibody (eBioscience) at 4 °C for 30 min [29]. Samples were then washed and resuspended in PBS for flow cytometric analysis. Analysis gates were set on the live lymphocyte population based on forward and side scatter characteristics. All flow cytometric analysis was performed on a FACS Canto II flow cytometer (BD Biosciences). A total of 20,000 events were acquired and analyzed using FlowJo software (Tree Star, Ashland, OR). Cultured autologous tumor cells were washed, pelleted, and lysed in RIPA buffer (25 mM Tris–HCl, 0.1% SDS, 1% Triton X-100, 1% sodiumdeoxycholate, 0.

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