Anti mouse igg2a b microbeads

All procedures were carried out as previously described [2 (link)] in accordance with Home Office Regulations (UK) and the UK Coordinating Committee on Cancer Research guidelines and by locally approved protocols (Home Office Project licence no. 40-3306). In some instances, CDX were passaged following disaggregation using a human tumour dissociation kit (Miltenyi Biotech) following the manufacturer's instructions. Dead cells were removed from the disaggregated tumour with a dead cell removal kit (Miltenyi Biotech) following the manufacturer's instructions. Murine cells were removed by mixing 20 µl anti-mouse IgG2a+b microbeads (Miltenyi Biotech), 10 µl anti-mouse MHC Class I antibody (eBioscience), and 500 µl binding buffer (Miltenyi Biotech), incubating at 4°C for 30 min, mixing with disaggregated tumour cells and incubating at room temperature for 15 min. Cell–bead mixture was then applied to an LS column (Miltenyi Biotech) in a MidiMACS separator (Miltenyi Biotech), the flow through collected, the column washed with 4 × 3 ml binding buffer, and the flow through and wash containing the human cells combined. Disaggregated cells were collected by centrifugation, resuspended in 10% DMSO in fetal bovine serum (Biowest) and stored at −80°C or in liquid nitrogen before re-implantation.

On day 14 of differentiation, cells were detached from culture vessels as described above. Cells were harvested in basal media with 10 µM Y-27632 and spun at 216× g for 3 min at 4 °C. After straining and counting, up to 15 × 10⁶ cells were first incubated in MACS buffer (PBS w/o (Gibco, Billings, MT, USA), 1.0% BSA (Sigma Aldrich, Saint Louis, MO, USA), 2 mM EDTA (Sigma Aldrich, Saint Louis, MO, USA) with FcR blocking (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min at 4 °C and mouse anti-human CPM antibody (FUJIFILM Wako; 1:200 dilution) for additional 20 min at 4 °C. Unbound antibody was removed by washing once with MACS buffer before cells were incubated in anti-mouse IgG 2a+b microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for 20 min at 4 °C. After washing once with MACS buffer, cells were separated using the QuadroMACS Separator (Miltenyi Biotec, Bergisch Gladbach, Germany). NKX2.1 flow cytometry analysis was used to determine the proportion of lung progenitor cells before and after sorting.