Lung epithelial cells were isolated as previously described13 (link), with the following modifications. After installation with agarose and subsequent hardening by a brief incubation on ice, each lobe was cut away from the mainstem bronchi. The proximal-most ¼ of each lobe surrounding the bronchi was then cut away to minimize the inclusion of basal cells in the cell preparation, and the previous protocol was followed from this point on.
For FACS analysis, single cell preparations were incubated 30–45 minutes at 4°C with the following primary antibodies: PE, Alexa Fluor 488, or BV421-conjugated rat anti-mouse EpCAM (1:500; Biolegend, G8.8), Alexa Fluor 647 or PE-conjugated rat anti-mouse integrin β4 (1:75; BD, 450-9D), Alexa 647-conjugated CD200 (1:100, Biolegend, OX-90), and PE/Cy7 conjugated CD14 (1:100, Biolegend, Sa14-2). Antibody incubations were done in DMEM (without phenol red) + 2% FBS and cells were washed twice with PBS after antibody incubations. Sorting and analysis was performed on BD FACS Aria cytometers.
For FACS analysis, single cell preparations were incubated 30–45 minutes at 4°C with the following primary antibodies: PE, Alexa Fluor 488, or BV421-conjugated rat anti-mouse EpCAM (1:500; Biolegend, G8.8), Alexa Fluor 647 or PE-conjugated rat anti-mouse integrin β4 (1:75; BD, 450-9D), Alexa 647-conjugated CD200 (1:100, Biolegend, OX-90), and PE/Cy7 conjugated CD14 (1:100, Biolegend, Sa14-2). Antibody incubations were done in DMEM (without phenol red) + 2% FBS and cells were washed twice with PBS after antibody incubations. Sorting and analysis was performed on BD FACS Aria cytometers.