The monoclonal Abs that were used to stain cells in the flow cytometry analyses were as follows: anti-mouse CD8a (53-6.7), anti-mouse CD4 (GK1.5), anti-mouse CD44 (IM7), anti-mouse 62L (MEL-14), anti-mouse KLRG1 (2F1/KLRG1), anti-mouse IL-7R (A7R34), anti-mouse IL-2 (JE56-5H4), anti-mouse IFN-γ (XMG1.2), and phycoerythrin (PE) donkey anti-rabbit immunoglobulin G (IgG) (Poly4064) and PE anti-mouse Ki-67 (16A8). All these antibodies were purchased from BioLegend. The fluorescein isothiocyanate (FITC) BrdU Flow Kit and 7-aminoactinomycin D (7AAD) were purchased from BD Biosciences.
For intracellular cytokine staining, splenocytes and lymph node cells were stimulated for 4 hours in vitro with phorbol 12-myristate 13-acetate (PMA)/ionomycin in the presence of monensin and brefeldin A. The stimulated cells were fixed and permeabilized using a Fixation/Permeabilization Solution kit (BioLegend). Cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter). CytExpert software was used for data analysis. Naïve (CD62L+CD44−) and effector (CD62L−CD44+) CD8+ T cells were sorted with BD FACSAria (BD Biosciences).
For intracellular cytokine staining, splenocytes and lymph node cells were stimulated for 4 hours in vitro with phorbol 12-myristate 13-acetate (PMA)/ionomycin in the presence of monensin and brefeldin A. The stimulated cells were fixed and permeabilized using a Fixation/Permeabilization Solution kit (BioLegend). Cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter). CytExpert software was used for data analysis. Naïve (CD62L+CD44−) and effector (CD62L−CD44+) CD8+ T cells were sorted with BD FACSAria (BD Biosciences).