Moflo sx

Utricles from Lgr5-EGFP-CreERT2+ and Plp1-tdTomato+ mice were cultured with 1 mM neomycin for 24 h to damage the HCs and to re-induce the expression of Lgr5. To obtain a single-cell suspension, all tissues were gently removed from the Petri dishes (Greiner Bio-one) using fine forceps and subjected to 0.125% trypsin at 37°C for 10 min. Soybean trypsin inhibitor was added to stop the enzymatic reaction. The cell suspension was carefully triturated with plastic pipette tips (epTIPS Filter 20–300 μl; Eppendorf), and the cells were passed through a 40 μm cell filter (Greiner Bio-one) and collected in an Eppendorf tube. Dissociated cells were sorted on a MoFlo^® SX fluorescence activated cell sorting (FACS) cytometer (Beckman Coulter, CA, USA) using the channels for GFP and tdTomato, and the positive fractions were collected for further experiments.

For FACS sorting, lungs from WT or Sftpc-CreER; tdTomato⁺ mice were collected at 6 to 8 weeks of age and processed into a single-cell suspension using dispase, DNase I and collagenase I, as previously described [15 (link)]. The AEC2 population (Sftpc⁺ AEC2 cells) was isolated from the lungs of 6–8-week-old Sftpc-CreER; tdTomato⁺ mice 5 days after induction with 200 μg/g body weight tamoxifen. Krt5⁺ AEC2s and Krt5⁻ AEC2s were sorted by MoFlo SX (Beckman Coulter, Miami FL). Data were analyzed in Summit 5.2 (Beckman). For FACS analysis and sorting of mouse, the following antibodies were used: CD45 (Biolegend, 103,116, 1:100), LysoTracker (Invitrogen, L7526, 1:14000), EpCAM (Biolegend, 118,206, 1:100), Krt5 (Abcam, ab193895, 1:300), and Edu (50 mg/kg, E10187, Life Technologies). For the FACS analysis and sorting of patients, the following antibodies were used: LysoTracker (Invitrogen, #L7526, 1:14000), CD45 (Biolegend, #368,530, 1:1000), KRT5 (ABcam, #ab193895, 1:1000); EpCAM (Biolegend, #324,220, 1:1000).