Epcam apc cy7

Colons were harvested from aged mice (14–18 wk) and placed in cold 1× PBS on ice. Colons were cleaned of feces by flushing with cold 1× PBS. The colon was then cut open longitudinally to expose the epithelial layer and placed in 20 ml 2 mM EDTA at 37°C with vigorous shaking for 30 min. The supernatant was then transferred to a clean tube, and cells were pelleted at 300 g for 10 min. Cell pellets were then resuspended in 4 ml of 1 mg/ml Collagenase-Dispase cocktail (269638; Roche), 2% FBS, and 10 µg/ml DNase in RPMI for 10 min with occasional agitation. Single-cell suspensions were washed with MACS buffer followed by centrifugation at 340 g for 5 min at 4°C. Cells were then stained with specific surface antibodies: EpCAM APC-Cy7 (118218; Biolegend) and CD45 PE-Cy7 (103114; Biolegend). For intracellular staining, cells were fixed and permeabilized using BD Cytifix/Cytoperm (554714; BD Biosciences), then stained with anti-Chga (ab15160; Abcam) followed by secondary stain with goat anti-rabbit IgG conjugated to AF555 (ab150078; Abcam). DAPI (D9542; Sigma-Aldrich) was added before acquisition to visualize nuclei. Cells were resuspended in 20–30 µl MACS buffer, acquired on an ImageStreamX Mark II flow cytometer (for details, see above), and analyzed using IDEAS 6.2 software (Amnis Corp.).