B220 fitc ra3 6b2

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.

Recipient mice were irradiated at 800rad in an X-ray irradiator and placed on antibiotics by diluting 6mL of Pediatric Suspension Cherry Flavor (NDC 65862-496-47) into 250mL water bottles. Approximately 16 hours later, bone marrow was harvested from donor mice by flushing the marrow from femurs, tibias, and humeruses with 2.5% FBS in PBS. The cell suspension was run through 70μm cell strainers and red blood cells lysed by treating with ACK lysis buffer for 2min at room temperature before lysis was stopped upon addition of further 2.5% FBS in PBS. Cell suspensions were washed twice with PBS before being suspended in PBS for injection via tail vein. Recipient mice received 6x10⁶ donor bone marrow cells in 0.2mL total volume. Mice were kept on antibiotics for 6 weeks. To assess chimerism via congenic markers, blood was collected via retro-orbital bleeds from recipient mice at week 8, RBCs lysed with ACK lysis buffer as described above, and samples stained for flow cytometry. The markers used were CD8-BV785 (53–6.7, BioLegend), CD4-BV786 (RM4-5, BD Biosciences), CD11b-FITC (M1/70, Tonbo), CD11c-PECy7 (N418, Biolegend), B220-FITC (RA3-6B2, eBioscience), CD45.1-PerCPCy5.5 (A20, BioLegend), and CD45.2-PE (104, eBioscience).