Pacific blue anti mouse cd3

Carprofen (Rimadyl^® Injectable, 50 mg/mL) was purchased from Patterson Companies (Saint Paul, MN, USA). Pacific Blue™ anti-mouse CD3, FITC anti-mouse CD4, PerCP anti-mouse CD8a, PE anti-mouse/human CD11b, APC/Cy7 anti-mouse Ly-6G/Ly-6C (Gr-1), PE/Cy7 anti-mouse CD62L, APC anti-mouse/human CD44, APC anti-mouse CD25, Biolegend PE anti-mouse/rat/human FOXP3, PerCP anti-mouse CD11c, PE/Cy7 anti-mouse CD86, FITC anti-mouse I-A/I-E, and APC anti-mouse CD40 were purchased from Biolegend (San Diego, CA, USA). Mouse tumor dissociation kit (No. 130-096-730) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Single-cell suspensions of splenocytes (1 x 10⁶ cells/well) were first stained for 45 min at 4°C with H-2Kb (SIINFEKL) dextramers conjugated to phycoerythrin (PE) (Immudex, Copenhagen, Denmark). Cells were then incubated with Pacific Blue^™ anti-mouse CD3 (Biolegend, San Diego, CA, US), anti-mouse CD8a APC-eFluor^® 780 (eBioscience, San Diego, CA, US) and anti-mouse CD62L (L-Selectin) Alexa Fluor^® 700 (eBioscience) antibodies for 30 min at 4°C. After fixation and permeabilization using Fix/Perm buffer (eBioscience), cells were intracellularly stained with anti-human/mouse T-bet PE-Cyanine7 (eBioscience) and Alexa Fluor^® 647 anti-human/mouse Granzyme B (Biolegend) antibodies for 1 h at 4°C. Flow cytometry analysis was performed using a BD LSRII flow cytometer and BD FACSDIVA software (Becton Dickinson, NJ, US). The results were analyzed using FLOWJO software.