Anti cd8 percp cy5.5 clone 53 6 7

Splenocytes from immunized mice were plated in a 96-well plate at 10⁶/well in the presence of DMSO or 10 μg/mL GUCY2C _254-262 peptide. Cells were incubated at 37 °C for 1 h, a protein transport inhibitor cocktail (eBioscience) was added, and splenocytes were incubated for an additional 5 h at 37 °C. Cells were then stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen), anti-CD8-PerCP-Cy5.5 (clone 53-6.7; BD Biosciences # 551162; 1:200 dilution), and anti-CD19-BV510 (clone 6D5; Biolegend # 115545; 1:200 dilution). A BD Cytofix/Cytoperm Kit (BD Biosciences) was used for permeabilization and intracellular cytokine staining using anti-IFNγ-PE-CF594 (clone XMG1.2; BD Biosciences # 562333; 1:200 dilution), anti-TNFα-PE-Cy7 (clone MP6-XT22, BD Biosciences # 561041; 1:200 dilution), and anti-MIP1α-APC (clone 39624; R&D Systems # IC450A; 1:200 dilution). Cells were fixed in 2% paraformaldehyde and analyzed on a BD LSR II flow cytometer. Analyses were performed using FlowJo software (TreeStar).

For intracellular cytokine staining (ICS), ACK lysis buffer-treated whole-blood PBMCs were incubated for 5.5 to 6 h in the presence of a peptide pool representing the antigen of interest at individual peptide concentrations of 5 μg/ml. Golgi-Plug (BD Biosciences) (1 μl/ml) and anti-CD107a PE (clone eBio/D4B) at a final dilution of 1:200 were also included. Phenotypic analysis of CD8⁺ and CD4⁺ T cells was performed by staining PBMCs using the following antibody clones: anti-CD8 PerCP-Cy5.5 (clone 53-6.7) and eFluor 650-coupled anti-CD4 (GK1.5) and anti-IFN-γ APC (XMG1.2) (BD Biosciences). Also, the surface staining of liver-resident lymphocytes was performed using the following antibodies: anti-NK1.1 FITC, anti-CD3 Alexa Fluor 700, anti-CD69 PE-Cy 7, anti-CD8 eFluor 450 (BD Biosciences). The liver-resident invariant natural killer T cells (iNKT cells) were stained with CD1d tetramer conjugated to PE. Nonspecific binding of antibodies was prevented by incubating with anti-CD16/CD32 Fcγ III/II receptor prior to staining. Flow cytometric analyses were performed using an LSRII instrument. Data were analyzed using either FACSDiva or FlowJo software. Analysis of multifunctional CD8⁺ T-cell responses was performed using Boolean analysis in FlowJo software, Pestle, and SPICE 4.0 kindly provided by M. Roederer (National Institutes of Health, Bethesda, MD).