Characterization of 4-1BB Expression on Lung Cells

Single-cell suspensions of lungs were prepared from mice as previously described (22 (link)). To reduce non-specific antibody binding, cells were incubated with a Fc receptor block (BD Pharmingen, San Jose, CA, USA). Then lung single-cell suspensions were stained with antibodies to CD45 (BD Pharmingen), F4/80 (Miltenyi Biotech, Bergisch Gladbach, Germany), CD11c (BD Pharmingen), CD137 (eBioscience™, San Diego, CA, USA), CD137L (eBioscience™), CD4 (BD Pharmingen), CD44 (BD Pharmingen), and CD62L (BD Pharmingen). For MH-S cells, the cell surface was stained by CD137 or CD137L antibodies after Fc blocking. Isotype-matched mAb were used as negative controls. A FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo V10 software were used to analyze the expression of 4-1BB on lung cells.