Cd45r b220 pe vio770

Individual fetal lungs were harvested and homogenized in 2% fetal bovine serum (FBS) in PBS using a 70 μm cell strainer. The resulting single cell suspension was pelleted and resuspended in 2% FBS in PBS and incubated with a 1:1000 dilution of FcR Blocking reagent (Cat # 130-059-901, Miltenyi Biotec Inc., Auburn, CA) for 30 minutes. Fetal lungs were subsequently stained for CD326 (EpCAM)-Super Bright 436 (Cat # 62-5791-82, Invitrogen, Carlsbad, CA), CD31(PECAM-1)-APC (Cat# 17-0311-82, Invitrogen, Carlsbad, CA), and CD45R(B220)-PE-Vio770 (Cat# 130-102-817, Miltenyi Biotec Inc., Auburn, CA). Aliquots of lung cells were also stained using SYTOX Orange Dead Cell Stain (Cat# S3486, Invitrogen, Carlsbad, CA). Cells were immediately analyzed by flow cytometry (BD LSRII 5-UV) and cell analysis was performed using FlowJo (Version 10.6.1, Ashland, OR). Cell populations were defined as follows: epithelial, CD326+CD31−CD45−; endothelial, CD326−CD31+CD45−; hematopoietic lineage, CD31−CD45+ as previously described (Supplementary Figure 1).[32 (link)]