Mouse pandc enrichment kit

CD8⁺ T cells from P14 or WT mice were magnetically sorted from spleens after mechanical dissociation and red blood cell lysis using the EasySep mouse CD8⁺ T-cell enrichment kit (Stemcell). Purified T cells were maintained overnight in complete medium at a concentration of 1 x 10⁶ cells/ml before starting coculture with DCs. Purity was assessed by flow cytometry and was > 90%.
For DC purification, spleens were mechanically dissociated and treated with 16 mg/ml collagenase and 10 mg/ml DNAse at 37°C for 30 min and then filtered through a 40-µm cell strainer. Negative cell sorting was performed using the mouse PanDC Enrichment kit (Miltenyi Biotec) according to the manufacturer’s instructions. DCs were conserved overnight in PBS, 1% FCS, and 2 mM EDTA at 4°C before use. Purity of the DC cell suspension was assessed by flow cytometry and reached 70% for the transcriptomic experiments.

The C5BL/6 spleen cells from wild type untreated mice were cut into pieces with scalpel blades, and then digested with collagenase solution at 37C for 30 min on a shaker. The digested spleen cells were filtered with a cell strainer (70μm), and then RBCs were lysed with lysis buffer. For sorting, Miltenyi Mouse Pan-DC enrichment kit was used to enriched CD11c⁺ cells by magnetic beads labeling and purification on a LS column. For sorting DC cell subsets, the enriched CD11c⁺ cells were stained with the following monoclonal antibodies; CD11c, B220, MHCII, CD8a, and CD172a. The sorted DC subsets (DC1a (Pop 1), DC1b (Pop 2), DC2 (Pop 3) used for antigen (Ova) presentation to OT-I T cells, were irradiated (3,000 rads) from a ¹³⁷Cs source (J.L. Shephard & Associates, San Fernando; CA).