Flow cytometry
Following digestion with Liberase (385µg/mL PBS; SigmaAldrich; Glasgow, Scotland, UK) and DNaseI (0.5mg/mL; SigmaAldrich; Glasgow, Scotland, UK) and red blood cell lysis (Hybrimax buffer; SigmaAldrich; Glasgow, Scotland, UK) 2x106 lung cells per panel were stained with LIVE/DEAD fixable near-IR dead cell kit (ThermoFisher Scientific; Waltham, MA, USA). The following anti-mouse antibodies were used for immune cell characterisation; Brilliant Violet 510 CD11c (N418; Biolegend; London, UK), Brilliant Violet 570 CD11b (M1/70; Biolegend; London, UK), Brilliant Violet 605 CD45 (30-F11; Biolegend; London, UK), AlexaFluor 647 CD64 (X54-5/7.1; Biolegend; London, UK), AlexaFluor 700 Ly6C (HK1.4; Biolegend; London, UK), phycoerythrin Siglec-F (E50-2440; BD Biosciences; Wokingham, Berkshire, UK), eFluor 450 Ly6G (1A8; ThermoFisher Scientific; Waltham, MA, USA). Fc-receptor binding was blocked using anti-mouse CD16/CD32 antibody (2.4G2) (BD Biosciences; Wokingham, Berkshire, UK).
Red blood cell were lysed (RBC lysis buffer; Biolegend; London, UK) and 50 µl per panel was stained with dead cell kit as above and the following antibodies used; Lineage dump (Brilliant Violet 421 Siglec F (E50-2440; BD Biosciences), Pacific Blue CD3 (17A2; Biolegend; London, UK), CD19 (6D5; Biolegend; London, UK), and CD49b (PK136; Biolegend; London, UK)), Brilliant Violet 510 Ly6G (1A8; Biolegend; London, UK), Brilliant Violet 605 CD45 (30-F11; Biolegend; London, UK), Brilliant Violet 650 CD11b (M1/70; Biolegend; London, UK), phycoerythrin Treml4 (16E5; Biolegend; London, UK), allophycocyanin CD115 (AFS98; Biolegend; London, UK), AlexaFluor 700 Ly6C (HK1.4; Biolegend; London, UK). Fc-receptor binding was blocked as above.
2x106 bone marrow cells were stained with dead cell kit as above, lineage dump (as above plus Pacific Blue Ly6G (1A8; Biolegend; London, UK), TER-119 (TER-119; Biolegend; London, UK), CD11c (N418; Biolegend; London, UK)), BV711 CD16/CD32 (93; Biolegend; London, UK), PE-Cy7 CD117 (2B8; Biolegend; London, UK), allophycocyanin CD115 (AFS98; Biolegend; London, UK) and AlexaFluor 700 Ly6C (HK1.4; Biolegend; London, UK). Fc-receptor binding was blocked using purified mouse serum (M5905; SigmaAldrich; Glasgow, Scotland, UK).
Flow cytometry samples were analysed with an Aurora cytometer (Cytek; Amsterdam, The Netherlands) and analysis performed with FCS Express 7 (De Novo Software; Pasadena, CA, USA).