[25] Preparation of individual smooth muscle cells from the stomach of Bufo marinus

Publisher Summary This chapter describes a method for isolation of single smooth muscle cells from the stomach of the toad Bufo marinus. For preparation sections of muscle layer sliced on Stadie-Riggs slicer to 0.5 mm thickness, trypsin and collagenase is added subsequently during incubation. The first and second incubations following the initial trypsin/collagenase exposure usually yield a population of smooth muscle cells that contain the highest percentage of relaxed, fully extended cells. As a further test of the physiological integrity of the isolated smooth muscle cells, the responsiveness is tested to an excitatory stimulus. This can be done in a quantitative manner by determining the doseresponse curve to carbachol utilizing a Coulter counter. The cells are then transferred from the dilute collagenase solution in which they were isolated to another solution by sedimenting them at low speed in a centrifuge for 20 min. After sedimentation at low speed, the cells may be resuspended in APS or various buffers with modified ionic composition useful for electrophysiological or ion flux studies.