Isolation of Murine Immune Cells

Spleen and lymph nodes were harvested from 6–12-wk-old mice. Single-cell suspension was obtained by dissociation using sterile frosted slides and passing through a 70-µm cell strainer. Red blood cell lysis was performed as needed. Naive CD4 T cells were isolated according to MojoSort kit protocol (BioLegend). The purity of naive CD4 T cells was constantly monitored and maintained at >95% CD4⁺MHCII⁻CD62L⁺CD44⁻. Splenic DCs were isolated from the spleen of a mouse injected with B16-FLT3L melanoma. Splenocytes were blocked with Fc block (anti-CD32/CD16) and stained with CD11c-biotin (BioLegend), then with anti-Biotin beads (Miltenyi), and isolated using AutoMacs magnetic selection (Miltenyi). The purity of isolated splenic DCs was maintained at >98% CD11c⁺. For all experiments, DC donor mice and naive CD4 T cell donor mice were age and sex matched. B cells were isolated from sex- and age-matched naive mouse spleen by isolating the CD19⁺ population using CD19-biotin (BD) and AutoMacs (Miltenyi). LP lymphocytes (LPLs) were isolated as previously described (Hu et al., 2011 (link)).