Cd117 c kit

The immunophenotype of the long-term cultures of limbal explants containing outgrowing cells was determined by flow cytometry. FITC, R-phycoerythrin (PE) and allophycocyanin (APC) conjugated antibodies were used to measure the expression of CD34, CD44, (all from BD Biosciences, San Jose, CA, USA); CD31, CD47,CD90/Thy-1, CD117/c-kit, CD146/MCAM, CD166/ALCAM, CXCR4 (all from R&D Systems, Minneapolis, MN, USA) (for further details refer to S2 Table). Samples were measured by FACS Calibur flow cytometer (BD Biosciences Immunocytometry Systems) and data were analyzed using Flowing Software 2.5 (PerttuTerho, Turku Centre for Biotechnology, University of Turku, Finland). For comparison, surface marker expression of short-term (2 weeks) cultivated LESCs was used–the isolation and cultivation of such LESCs were based on a previous study[8 (link)].

Jejunum and colon tissues were analyzed by whole mount and cryostat section staining using confocal microscopy as previously described.^{32 (link)} Cell-specific primary antibodies against the following antigens were used: anti-MYH11 (1 : 800, Alfa Aesar, Ward Hill, MA, USA) for SMCs, CD117/c-kit (1 : 20, R&D Systems, Minneapolis, MN, USA) for ICCs or PGP9.5 (1 : 1000, UltraClone Limited, Wellow, UK) for enteric nerve system. For histological analysis, jejunum tissues were dehydrated, embedded in paraffin, cut into 4 μm-thick coronal sections, rehydrated and stained with H&E. Images were collected using an Olympus FV1000 confocal laser scanning microscope with Fluoview FV10-ASW 3.1 Viewer software (Olympus, Tokyo, Japan) or the iScan Coreo scanner (Ventana Medical Systems, Tucson, AZ, USA).