ac960412j_si_031.jpg (8.24 kB)

Monitoring Exocytosis and Release from Individual Mast Cells by Capillary Electrophoresis with Laser-Induced Native Fluorescence Detection

Download (8.24 kB)
posted on 01.09.1996, 00:00 by Sheri J. Lillard, Edward S. Yeung, Michael A. McCloskey
The complex temporal evolution of on-column exocytotic release of serotonin and proteins from individual rat peritoneal mast cells (RPMCs) was monitored by using capillary electrophoresis. Laser-induced native fluorescence detection with 275-nm excitation was used, and a detection limit of 1.7 amol (S/N = 3; rms) was obtained for serotonin. A physiological running buffer was used to ensure that the cell remained viable throughout. The secretagogue was polymyxin B sulfate (Pmx). Following the injection of a single mast cell into the capillary, electromigration of Pmx toward and past the cell induced degranulation and release of serotonin. The time course of release was registered in the electropherograms with subsecond resolution. Subsequent introduction of SDS caused the cell to lyse completely and allowed the residual serotonin to be quantified. The average amount of serotonin observed per RPMC was 1.6 ± 0.6 fmol; the average percentage of serotonin released was 28 ± 14%. Events that are consistent with released serotonin from single submicrometer granules (250 aL each) were evident, each of which contained an average amount of 5.9 ± 3 amol.