Anti cd326 epcam pe cy7

Whole lungs were harvested from euthanized mice (Cracd WT or Cracd KO) after perfusing 10 ml of cold phosphate-buffered saline (PBS) into the right ventricle. The lung was digested in Leibovitz’s medium (Invitrogen) with 2 mg/mL Collagenase Type I (Worthington), 2 mg/mL Elastase (Worthington), and 2 mg/mL DNase I (Worthington) at 37 °C for 45 min. The tissue was triturated with a pipet every 15 min of digestion until homogenous. The digestion was stopped with FBS (Invitrogen) to a final concentration of 20%. The cells were filtered with a 70 μm cell strainer (Falcon) and spun down at 5,000 r/min for 1 min. The cell pellet was resuspended in red blood cell lysing buffer (Sigma) for 3 min, spun down at 5,000 r/min for 1 min, and washed with 1 mL ice-cold Leibovitz’s medium with 10% FBS. In single-cell RNA sequencing (scRNA-seq), digested lung cells were resuspended in 400 μl of buffer with 5 μl of anti-CD31-FITC (BD Biosciences, CA, USA), 5 μl of anti-CD45-APC (BD Biosciences), and 5 μl of anti-CD326 (EpCAM)-PE-Cy7 (Biolegend) and incubated for 30 min at 4 °C. Cells were then washed twice, followed by sorting of the epithelial cells (EpCAM+ / CD31− / CD45−) by fluorescence-activated cell sorting at the Cytometry and Cell Sorting Core at the Baylor College of Medicine.

Lungs were prepared and cells were flow sorted as previously described (Lee et al., 2014 (link)). Briefly, mice were anesthetized with avertin overdose. Lungs were perfused with cold PBS followed by intratracheal instillation of 2 mL dispase (Corning). Lungs were placed on ice, minced, and incubated in 0.0025% DNase (Sigma-Aldrich) and 100 mg/mL collagenase/dispase (Roche) in PBS for 45 min at 37°C. Cells were then sequentially filtered through 100- and 40-μm cell strainers (Falcon) and centrifuged at 1000 rpm for 5 min at 4°C. Cells were resuspended in red blood cell lysis buffer (0.15 M NH4Cl, 10mM KHCO3,0.1 mM EDTA) for 90 s at room temperature, followed by addition of DMEM (Gibco) and FBS. Cells were centrifuged at 1000 rpm for 5 min at 4°C and then resuspended in PBS/10% FBS for further staining. The following antibodies were used: anti-CD31 APC, anti-CD45 APC, anti-Ly-6A/E (SCA1) APC/Cy7 (all Thermo Fisher Scientific), anti-CD326 (EpCAM) PE/Cy7 (Biolegend) (all 1:100). DAPI (Sigma-Aldrich) was used to eliminate dead cells. Single stain controls and fluorescence minus one (FMO) controls were included for each experiment. FACS was performed on a FACSAria II and analysis was done on FlowJo (BD).