Fig. 1. Immunofluorescent labelling and purification of interstitial cells of Cajal (ICC) in human gastric muscle tissue. (A) Workflow used to investigate primary human ICC from sleeve gastrectomy samples. (B) Co-localization of calcium-activated chloride channel anoctamin-1 (ANO1) (green; left) and receptor tyrosine kinase (KIT) (red; middle) in sectioned human gastric tissue. Merged image (right) shows ANO1+/KIT+ labelled ICC (arrow) with DAPI staining indicating cell nuclei (blue). Scale bar = 50 µm. (C) Representative flow cytometry plot showing key cell populations: negative cells (NEG; KIT–/CD45–/CD11B–), hematopoietic cells (HCs; KIT–/CD45+/CD11B+), mast cells (MCs; KIThigh/CD45+/CD11B+), and ICC (KITlow/CD45–/CD11B–). (D) ICC frequency and sorted ICC numbers. Each dot represents a patient sample. (E) Real-time polymerase chain reaction data for the ICC population versus the unsorted population using cell-type markers (ANO1, CACNA1H, KIT, CD68, CPA3, UCHL1, DES, and ACTG2; data normalized to GAPDH and expressed as mean fold change ± standard error of the mean relative to unsorted cells––dotted line). Significant differences compared to unsorted cells: *P < 0.05, **P < 0.01, ***P < 0.001. (F, G) Merged (bright-field, ANO1 fluorescence in green and DAPI nuclear stain in blue) images of purified human gastric ICC (F) and NEG (G) after 2 days in culture. Scale bar = 10 µm.
© J Neurogastroenterol Motil
