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Immunomagnetic enrichment of interstitial cells of Cajal

¹Department of Physiology and Cell Biology and ²Cytometry Center, University of Nevada, Reno 89557; and ³Sierra Cytometry, Reno, Nevada 89509

Submitted 1 July 2003 ; accepted in final form 7 October 2003

Disruptions of networks of interstitial cells of Cajal (ICC), gastrointestinal pacemakers and mediators of neurotransmission, can lead to disordered phasic contractions and peristalsis by reducing and uncoupling electrical slow waves. However, detailed analysis of the ICC network behavior has been hampered by their scarcity, limited accessibility in intact tissues, and contamination with other cell types in culture. Our goal was to develop a simple technique to purify ICC from murine gastrointestinal muscles for functional studies. We identified ICC in live small intestinal muscles or primary cell cultures by Kit immunoreactivity using fluorescent antibodies. Because this technique also labels resident macrophages nonspecifically, parallel studies were performed in which nonfluorescent Kit antibodies and macrophages labeled with fluorescent dextran were used for subtractive analysis of ICC. In both groups, Kit-positive cells were tagged with superparamagnetic antibodies and sorted on magnetic columns. Efficacy was assessed by flow cytometry. ICC enrichment from primary cultures and freshly dissociated tissues was 63-fold and 8-fold, respectively. Unlike the cells derived directly from tissues, cells sorted from cultures frequently yielded extensive, nearly homogenous ICC networks on reseeding. Monitoring oscillations in mitochondrial Ca²⁺ or membrane potential by imaging revealed spontaneous rhythmicity in these networks. Cells that did not bind to the columns yielded cultures that were depleted of ICC and dominated by smooth muscle cells. In conclusion, immunomagnetic sorting of primary cultures of ICC results in relatively homogenous, functional ICC networks. This technique is less suitable for obtaining ICC from freshly dispersed cells.

mouse; Kit; macrophage; pacemaking; fluorescent imaging

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