Figure 1. Schema for purification of primary CCSP positive cells from mouse lung.

A) Two month old FVB mice were euthanized by CO_2, lungs removed and lobes collected. After washing in PBS, lobes were minced on ice and incubated in a small cell culture dish with 3 mg/ml collagenase in PBS (total 5 ml) in a shaking platform for 1 hour at 37°C. The suspension was further disaggregated by trituration through a 19 gauge needle, with 5 ml of PBS, filtered through a 40 µm cell strainer and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, cells resuspended in red blood cell lysis buffer for 4 min re-plated into 10 cm culture dishes for recovery overnight (18 hrs). Surviving cells were adhering to the dish. After trypsinization and neutralization by 10% FBS media, cells were resuspended in PBS with 3% FBS, and stained with rabbit anti-CCSP antibody and FITC conjugated anti-rabbit secondary antibody. CCSP⁺ and CCSP⁻ cells were sorted with FACS Vantage SE cell sorter. B) Rabbit IgG was used as an isotype -matched negative control; CCSP⁺ population sorted with FACS Vantage SE cell sorter from dissociated lung tissue was 25.37%.