Sorting of ICC by FACS and identification of ICC.

<p>(A, D, G) Gates used for selecting cells with light scatter properties of live cells (LS cells). (B, E, H) Gates used for selecting cells that did not express macrophage markers (F4/80, CD11b) and pan leukocyte marker CD45 (PC5⁻ cells). (C, F, I) Gates used for separating ICC progenitors (c-kit⁺CD34⁺ cells) and mature ICC (c-kit⁺CD34⁻ cells). (A–C) Samples labeled with all markers. (D–F) FMO control without the c-kit antibody. (G–I) FMO control without the CD34 antibody. (J) Quantitative RT-PCR analysis of gene expression in the sorted ICC. Gene expression was normalized to GAPDH mRNA levels and expressed relative to the unsorted cells. Note the significant enrichment of ICC in the sorted ICC group. The expression of CD68 (a macrophage marker) and MCT (a mast cell marker) were not detected. However, MyHC (a smooth muscle cell marker) was observed but did not increase after sorting. (K) Immunohistochemical staining of cultured ICC using an antibody against ANO1 (red); nuclei were stained with DAPI (blue); negative control were treated in the same way but omitted the primary antibody.