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1 Physiology and Cell Biology, University of Nevada, Reno, Reno, Nevada, USA
2 Cytometry Center, University of Nevada, Reno, Reno, Nevada, USA; Sierra Cytometry, Reno, Nevada, USA
3 Cytometry Center, University of Nevada, Reno, Reno, Nevada, USA
4 Animal Biotechnology, University of Nevada, Reno, Reno, Nevada, USA
* To whom correspondence should be addressed. E-mail: tamas{at}physiology.unr.edu.
Interstitial cells of Cajal (ICC) in the gastrointestinal tract generate and propagate slow waves and mediate neuromuscular neurotransmission. Although damages to ICC have been described in several gastrointestinal motor disorders, analysis of their gene expression in health and disease has been problematic due to the difficulties in isolating these cells. Our goal was to develop techniques for large-scale purification of ICC. Murine ICC were identified in live gastrointestinal muscles with fluorescent Kit antibodies. Since this technique also labels resident macrophages nonspecifically, we attempted to separate ICC from these cells by fluorescence-activated cell sorting with or without immunomagnetic presorting. Efficacy and specificity of ICC purification were tested by quantitative RT-PCR of cell-specific markers. Fluorescence-based separation of small intestinal ICC from unlabeled cells and macrophages tagged with F4/80 antibodies yielded 30,000-40,000 cells and ~60-fold enrichment of c-kit mRNA. However, the macrophage marker CD68 was also enriched ~6-fold. Magnetic presorting of ICC did not significantly improve selectivity. After labeling contaminating cells with additional paramagnetic (anti-CD11b, -CD11c) and fluorescent antibodies (anti-CD11b) and depleting them by magnetic presorting, we harvested ~2,000-4,000 cells from single gastric corpus-antrum muscles and detected a ~30-fold increase in c-kit mRNA, no enrichment of mast cells, and a ~4-fold reduction of CD68 expression. Adding labeled anti-CD45 antibody to our cocktail further increased c-kit enrichment and eliminated mast cells and macrophages. Smooth muscle cells and myenteric neurons were also depleted. We conclude that immunofluorescence-based sorting can yield ICC in sufficiently high numbers and purity to permit detailed molecular analyses.
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