Evidence for Uptake of Vital Dye by Activated Rat Peritoneal Mast Cells: An in Vitro Imaging Study

Abstract

Uptake of material from surrounding medium by activated rat peritoneal mast cells (PMCs) was studied using in vitro peritoneal eluate cells, the vital fluorescent dye sulforhodamine B (SFRM-B), secretagogue compound 48/80, and an imaging technique. PMCs, which undergo different states of degranulation, are shown to possess the ability to take up (by endocytosis) SFRM-B in an activity-dependent manner. The endo-cytosed dye is incorporated in the granules and can be discharged into the medium when the cells are reactivated. Both the uptake and the discharge processes are calcium-dependent. The reactivity of mast cells to secretagogue is not altered by the application of the dye. SFRM-B, a negatively charged, nonspecific protein stain, displays greater photostability and less leakage than the positively charged acridine orange, and its fluorescence persists for hours, whereas acridine orange fluorescence fades within 1 min when exposed to ultraviolet illumination. The fluorescent image of the dye-loaded mast cells can be preserved overnight in a container at room temperature. SFRM-B elicits no detectable damaging influence on the activated afferent discharge of splanchnic afferent nerve fibers with mesenteric terminals. This enables the use of SFRM-B for studying the interactions between mesenteric afferent terminals and their surrounding mast cells.