Profiling of normal human colonic epithelial cells according to their PTK7 surface abundance

Human mucosa was collected from two different individuals undergoing colectomy. After treatment with EDTA, colonic crypts were isolated and further dis-aggregated using Dispase. Next, cells were stained using antibodies directed against the extracellular domain of EpCAM, PTK7, CD11, CD31, and CD45. Cells were analyzed and sorted via flow cytometry (BD Aria). After excluding non-epithelial cells (Epcam. CD11, CD31, and CD45 negative), the EpCAM positive fraction was divided into fractions expressing either high, medium, low, or negative levels of PTK7. Sorted cells were transferred to Trizol for RNA extraction and RNA was purified using the Qiagen RNA Mini Kit. Colonic crypts were isolated from normal human mucosa derived from individuals undergoing colectomy. Single cell fractions from these crypts were sorted and isolated RNA processed and hybridized to Affymetrix PrimeView Arrays