Epcam pe cy7

Xenografts were resected and disaggregated as previously described in Merlos-Suárez et al. (2011) (link) and Cortina et al. (2017) (link). Human epithelial cells from disaggregated PDX were first incubated 30 min at 4°C with 1:200 CD16/CD32 (mouse, Tonbo Biosciences, 70-0161-U500) to block free antibody binding sites and with 1:200 BV421-CD31 (rat, BD Biosciences, 562939cloneMEC13.3) and 1:200 BV421-CD45RB (rat, BD Biosciences, 562849clone16A) to stain for immune and endothelial mouse cells. After this period, 1:150 EPCAM-PeCy7 (human, eBioScience 25- 9326-42) or 1:100 EPCAM-APC-Vio770 (human, Miltenyl Biotec 130-101-161) was added and incubated for 1 h at 4°C. Mouse tumor cells from AKP xenografts were stained with 1:300 EPCAM-APC-Cy7 (mouse, Biolegends, 118217 cloneG8.8). DAPI (1 μg/ml) was added to distinguish live/dead cells. The cell suspension was analyzed with a BD Fusion FACS or Aria FACS.

Flow cytometry and cell sorting experiments were performed by using cell analyzer CytoFLEX LX (Beckman Coulter) and cell sorter MoFlo Astrios (Beckman Coulter). Lungs were harvested and digested into single cell suspensions using collagenase I, dispase, and DNase, as previously explained.^{6 (link),37 (link)} Red blood cells were removed with the ACK buffer and then stained with antibodies. The following antibodies were used for flow cytometry and cell sorting: EpCAM-PE-Cy7 (eBioscience, G8.8, 1:200), CD31-APC (eBioscience, 390, 1:200), and CD45-APC (eBioscience, 30-F11, 1:200). DAPI was used to gate out dead cells.

Litters of mice aged P0, P3, P6, or P10 were dissected, and their intestinal epithelium was isolated as described above. Upon washing of epithelial sheets in cold HBSS (containing Ca²⁺ and Mg²⁺), sheets were incubated in Epcam-PE/Cy7 (1:100; eBioscience) and APC-CD44 (1:25, BioLegend) for 1 h at 4°C with rotation. Sheets were washed in cold HBSS, then incubated in 0.8 mg/ml dispase II in HBSS at 37°C for 6-10 minutes with frequent shaking. A total of 1 ml FBS and 2 μl DNase I were added to each tube, and the cell solutions were passed through 70 μm cell strainers, followed by 40 μm cell strainers. Cells were collected by centrifugation at 2400 rpm for 5 min at 4°C. Cell pellets were washed with HBSS containing 10% FBS and spun at 2400 rpm for 5 min at 4°C. Pellets were then resuspended in DMEM, high glucose, containing Pen/Strep, B-27, 5% FBS, and 10 mM HEPES. Prior to FACs sorting, cells were run through a Celltrics 30 μm filter, and 1 ul Propidium Iodide was added to the cell suspension. Cells were collected into the same media as above using a BD DiVa cell sorter. Cells were pelleted, media was removed and cell pellets were stored at -80°C until RNA extraction.