Anti mouse igg1 rmg1 1 apc cy7

Human lung tissues were cleared by perfusion with cold PBS and cut into small pieces. Tissue was transferred to 10 ml of digestion buffers (2 ml of dispase II (Sigma), 100 μl of collagenase/dispase (Roche), 100 μl of 1% DNase I (Sigma), and 7.8 ml of PBS), followed by rotating incubation for 1hr at 37°C. The cells were then filtered through 40 μm strainers and centrifuged at 1500 rpm for 5min at 4°C. The cell pellet was resuspended in 1ml of RBC lysis buffer and lysed for 5 min at room temperature. 10ml basic F12 media (GIBCO) was added and 500μl of FBS (Hyclone) was slowly added in the bottom of tube. Cells were centrifuged at 1500rpm for 5 min at 4°C. The cell pellet was resuspended in PF10 buffer for further staining. The antibodies used were as follows: CD45 (2D1)-APC (BioLegend), CD31 (VM59)-APC (BioLegend), EpCAM (9C4)-FITC (BioLegend), HTII-280-mouse IgM (Terrace Biotech), Purified CD309/KDR (A16085H), anti-mouse IgG1 (RMG1-1)-APC-Cy7 (BioLegend), and anti-mouse IgM (Il/41)-PE (eBioscience). 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma) was used to eliminate dead cells. Flexible BD Influx^TM cell sorter were used for the sorting at Cambridge NIHR BRC Cell Phenotyping Hub and data were analysed with FlowJo software (Tree Star).