Isolation of Immune Cells from Murine Tissues

Cells from the aorta, spleen and paLN were isolated as described before^{19 (link)}. Briefly, Apoe^−/−Il27ra^+/− or Apoe^−/−Il27ra^−/− mice were euthanized by CO₂ inhalation. The aortas were perfused with 30 ml of PBS containing 2% heparin and isolated under the dissection microscope. Collected aortas, spleens or paLNs were cut into small pieces followed by digesting in 2 ml of enzymatic cocktail, containing 450 U/ml collagenase type I, 250 U/ml collagenase type XI, 120 U/ml hyaluronidase type I, 120 U/ml DNAse I (all enzymes from Sigma) in 1x HBSS and incubated in a shaker at 37 °C for 55 min. Obtained cell suspension was stained with following antibodies: CD45-PerCP (30-F11; BioLegend), CD11b-eFluor 450 (M1/70; eBioscience), CD11c-APC (N418; eBioscience), MHCII-Alexa Fluor 700 (M5/114.15.2; eBioscience), TCRβ-eFluor 780 (H57-597; eBioscience), CD69-PE-Cy7 (H1.2F3; eBioscience), B220-FITC (RA3-6B2; eBioscience), CD4-APC (GK1.5; eBioscience), CD8-PE (53-6.7; eBioscience) and LIVE/DEAD Yellow fixable dye (Invitrogen) and analyzed by flow cytometry (LSRII, BD Biosciences). Obtained data were analyzed using FlowJo software.