Cd3 v450

TKO-BLT-L mice were bled at 8- and 12-weeks post-surgery. Blood leukocytes were purified using RBC Lysis buffer (BioLegend, San Diego, CA, USA). Lung organoids were collected and digested using 60 mg/mL collagenase D (Roche, Basil, Switzerland) and 10 U/mL DNAse I for 1 h before passage through 70 mm filters. Leukocytes were then isolated using a 40%/70% Percoll gradient. Cells were incubated with anti-human Fc blocker (Thermo Fisher Scientific, Waltham, MA, USA) before antibody staining. Anti-mouse specific CD45, was used to exclude mouse leukocytes. Antibodies used included: CD3 V450, CD4 APC, CD8 APC-eFluor 780, CD14 PC7, CD16 FITC, CD19 PerCP-Cy5.5, CD45 V500, Live/Dead-PE-Texas Red, CD123 PE (eBioscience, San Diego, CA, USA), CD33 PE (Miltenyi, Bergisch Gladbach, Germany), CD56-AF700, CD11c-APC, HLA-DR and Lin-1 (CD3, CD14, CD16, CD19, CD20, CD56)-FITC (Biolegend). Data were acquired on a BD CytoFlex (Beckman Coulter, Brea, CA, USA) and analyzed with FlowJo Version 10.6 (BD Biosciences, Franklin Lakes, NJ, USA).