Enrichment and Analysis of Murine Immune Cells

Single cell suspensions were prepared from pooled murine spleens and lymph nodes (axillary, brachial, inguinal, cervical, mesenteric, pancreatic, and para-aortic) or thymuses by mechanical disruption. Cells were stained for 1 hour at room temperature with allophycocyanin-conjugated tetramers. In some experiments cells were also stained with anti-CXCR5 (2G8, BD) antibody or phycoerythrin-conjugated tetramers. Tetramer-binding cells were enriched on magnetized columns as described previously^{58 (link)}. For thymic epithelial cell and dendritic cell analysis, thymuses were harvested and enzymatically digested as previously described^{59 (link)}. Single cell suspensions were stained with a biotinylated antibody to CD11c (HL3, BD) and an allophycocyanin-labeled antibody to EpCAM (G8.8, Biolegend). Cells were incubated with anti-biotin and anti-allophycocyanin cocktails and magnetic beads (StemCell Technologies). Cells were enriched using the EasySep system according to the manufacturer’s instructions (StemCell Technologies).