The Ca signal from fura-2 loaded mast cells depends strongly on the method of dye-loading.

Abstract

The Ca concentration ([Ca2+]i) in single rat peritoneal mast cells was measured by means of the new fluorescent Ca-indicator dye fura-2. Dye-loaded cells were made to degranulate with either antigen or compound 48/80. In cells loaded with extracellularly applied, membrane-permeant fura-2 ester, degranulation was accompanied by a permanent loss of 40-60% of the fluorescence, but comparison of fluorescence at different wavelengths indicated no or only small changes in [Ca2+]i. When cells were loaded by microinjection of the impermeant potassium salt of the dye, degranulation resulted in no permanent loss of fluorescence, but instead was preceded by transient fluorescence changes that indicate a rapid, large and transient increase in [Ca2+]i. We suggest that ester-loaded fura-2 accumulates to a significant degree in the secretory granules and is lost from the cell during exocytosis.