Flow cytometric measurements were performed using the CytoFLEX S platform (Beckman Coulter Genomics, Brea, CA, USA). For extracellular staining of T cells, isolated splenocytes were incubated for 25 min with FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany); washed in PBS; incubated for 1 h at room temperature with CD3, CD4, and CD8 (Miltenyi Biotec) staining antibodies or H-2kb OVA Tetramer-SIINFEKL (MBL International, Woburn, MA, USA); washed again; and processed according to the manufacturer’s protocols.
Invitro-generated and activated DCs were washed in PBS, incubated for 30 min at room temperature with the 7-AAD viability dye (Sigma Life Sciences, Merck, Darmstadt, Germany), CD11c, CD80 (BioLegend, San Diego, CA, USA), CD86 (BD Biosciences, Franklin Lakes, NJ, USA), MHC class I (Invitrogen, Thermo Fisher Scientific), MHC class II (Miltenyi Biotec) DC lineage and activation marker antibodies, washed again, and processed according to the manufacturer’s protocols. The compensation was performed based on staining results from UltraComp beads (Thermo Fisher Scientific). All flow cytometry results were analyzed using Flow Jo (Ashland, OR, USA) software.
Invitro-generated and activated DCs were washed in PBS, incubated for 30 min at room temperature with the 7-AAD viability dye (Sigma Life Sciences, Merck, Darmstadt, Germany), CD11c, CD80 (BioLegend, San Diego, CA, USA), CD86 (BD Biosciences, Franklin Lakes, NJ, USA), MHC class I (Invitrogen, Thermo Fisher Scientific), MHC class II (Miltenyi Biotec) DC lineage and activation marker antibodies, washed again, and processed according to the manufacturer’s protocols. The compensation was performed based on staining results from UltraComp beads (Thermo Fisher Scientific). All flow cytometry results were analyzed using Flow Jo (Ashland, OR, USA) software.