Use of permeabilised mast cells to analyse regulated exocytosis

Publisher Summary This chapter describes a streptolysin-O (SL-O) prebind method for permeabilizing glass-attached primary rat peritoneal mast cells. This method is based on the temperature-independent binding of SL-O to the plasma membrane and the temperature-dependent polymerization of bound SL-O molecules required forming pores. Hexosaminidase, released from stimulated permeabilized cells into the supernatant, is assayed biochemically, while the remaining cells are processed for imaging. Thus, the functional and morphological research can be done in parallel. Immunostaining of mast cells poses specific problems due to the presence of highly charged secretory granules. Researchers need to use Sprague-Dawley rats. Retired breeders of either sex were used but other strains of various ages are also suitable. SL-O binds to the cells on ice, excess SL-O is removed by washing with a cold buffer, and cells are then permeabilized by the addition of warm buffer. Permeabilized cells are finally washed to remove freely soluble ions, nucleotides, and proteins before the addition of triggering solutions.