Anti cd8 mab

PBMC (2 × 10⁶ cells/mL) were stimulated with immobilized anti-CD3/anti-CD28 (each at 1 μg/mL) and IL-2 (100 U/mL) in the absence or presence of OECS conditioned neutrophils. After 24 h, 1 × protein transport inhibitor cocktail (Invitrogen) was added for the last 6 h. Following stimulation, cells were harvested, washed in PBS and incubated for 30 min at 4 °C in the darkness with the anti-CD8 mAb (Biolegend). Cells were then washed once in PBS containing 2% v/v FCS prior to fixation and permeabilization with 500 μL Cytofix/Cytoperm solution (Becton Dickinson) for 10 min at room temperature in darkness. Cells were washed once again and incubated with blocking buffer for 20 min at 4 °C in the darkness to prevent unspecific binding of the anti-cytokine monoclonal antibody. After another washing step, 5 μL anti-IFN-γ-FITC (Biolegend) were added and cells were incubated for 30 min at 4 °C in the darkness followed by a final washing step prior to measurement. Measurement was done using flow cytometer (Beckman CytoFLEX) using CytExpert Software.2.7.

Following the isolation of MedLNs, single cell suspensions were resuspended with 100ug/mL of LPS (Sigma Aldrich Cat. L2630-100MG) in BLAST medium (RPMI-1640 medium with 10% FCS, b-mercaptoethanol (50uM), L-glutamine, Sodium Pyruvate (2 mM) and PSA (2 mM)) and incubated for 3 hours at 37°C. All cells were treated for 1.5 hours at 37°C with Brefeldin A solution (1:1,000, Biolegend, Cat. 420601) and Monensin (1:1000, Biolegend, Cat. 420701). Cells were labeled with the following mAb mixture anti-CD45 mAb (Biolegend, Cat. 103108, clone 30-F11), anti-CD11c mAb (Biolegend, Cat. 117322, clone), anti-CD103 mAb (Biolegend, Cat. 121433, clone 2E7), anti-CD11b mAb (Biolegend, Cat. 101228, clone M1/70), anti-CD8 mAb (Biolegend, Cat. 100712, clone 53-6.7), fixed, permeabilized, and subjected to intracellular IL-6 staining with the anti-IL-6 mAb (Biolegend, Cat. 504504, clone MP5-20F3) using the Cytofix/Cytoperm kit (BD Biosciences, Cat. BD 554714).