Cd8α pe cy7

Splenic pan DCs were isolated from nine age-matched, female, littermate BALB/c mice and pooled per three mice for a total of three biological replicates. Pooled DCs were washed in flow buffer (PBS with 0.5% FBS), incubated with anti-mouse Fc Block (Thermo Fisher Scientific), and then stained with CD8α PE-Cy7 (Thermo Fisher; clone: 53–6.7), and CD24 Pacific Blue (Biolegend; clone: M1/69). CD8α+ and pre-cDC1 (CD24^high CD8α-) were sorted using FACSAria III (BD).

Transduction efficiency and TCR expression by T cells following transduction was measured via flow cytometry. The monoclonal antibodies against mouse CD8α-PE-Cy7 (BioLegend; clone:53-67), CD8α-PE-Cy7 (ThermoFisher; clone: 5H10), and CD8α-APC-R700 (BD Biosciences; clone: 53-67) were used. Cytokine production by T cell lines was analyzed with TNFα-MP6-XT22 (BioLegend; clone: MP6-XT22) and IFNγ-PE-Cy7 (BD Biosciences; clone XMG1.2) antibodies. TCR expression after transduction was evaluated based on tetramer staining. The following tetramers were used; Adpgk-tetramer-APC (ASMTNMELM-H-2-D^b), Adpgk-tetramer-PE (ASMTNMELM-H-2-D^b), Rpl18-tetramer-APC (KILTFDRL-H-2-K^b) and OvaI-tetramer-APC (SIINFEKL-H-2-K^b) (all MBL). TCR transduced T cells were stained for 30 min at 4°C. PBS containing 5% FBS and 5 mM EDTA was used as washing and staining buffer. Acquisition and analysis were performed on BD FACS CantoII and FlowJo softwares, respectively.