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).