Figure 1. Mucosa was dissected from ileum biopsies with and without radiation enteropathy(RE) and incubated in 0.2% collagenase Type II, 0.1% soybean trypsin inhibitor solution at 37°C to isolate primary subepithelial myofibroblasts. Cells were subcultured in FGM (Cambrex, Verviers, Belgium) used between passage 3 and passage 4. (a,b) After fixation and permeabilization, subsequent immunodetection of the -smooth muscle (sm) actin was performed as already described (CB) [13] (anti--sm actin; Sigma). Immunofluorescence experiments showed that subepithelial myofibroblasts derived from RE showed greater densities of stress fibres and nearly all cells were -sm actin positive. (c) Real-time reverse transcriptase polymerase chain reaction (RT-PCR) showed an increased connective tissue growth factor (CTGF) mRNA level in subepithelial myofibroblasts derived from RE vs subepithelial myofibroblasts derived from normal ileum. Values were normalized to GAPDH (glyceraldehyde-3-PDH) mRNA level. (d) Procollagen Type I enzyme-linked immunosorbent assay (ELISA) [procollagen Type I C-peptide (PIP) enzyme immunoassay; Takara Biomedicals, Cambrex, Belgium] showed an increased secretion of procollagen Type I in conditioned medium produced by RE subepithelial myofibroblasts vs normal subepithelial myofibroblasts.