Permeabilizing cells: some methods and applications for the study of intracellular processes.
The techniques described allow controlled permeabilization of plasma membranes from different types of cells for gaining access to the cell interior and enables one to control intracellular events. Most common techniques are electropermeabilization, permeabilization with mild non-ionic detergents such as saponin and digitonin and by pore-forming toxins, such as alpha-toxin and streptolysin O. Whereas electropermeabilization and alpha-toxin create small pores of approximately 2 nm, digitonin, saponin, and streptolysin O form bigger holes and therefore also allow the introduction of large molecules, such as enzymes and immunoglobulins. A disadvantage of the latter methods is the loss of cytosolic constituents which might be necessary for signal-transduction pathways in the cell. In secretory cells the main requirement for exocytosis appears to be Ca2+, which brings about the full response comparable to hormone effects in some cells (platelets), adrenal medullary cells, but not in all cells (pancreatic acinar cells). The nucleotide, anion, and cation requirements are different for different cell types and are probably intimately related to the cell-specific mechanisms involved in exocytosis such as regulation of ion channels and ion carriers, or the involvement of nucleotide-binding proteins. Since permeabilized cells are preparations intermediate between intact cells and isolated organelles, they offer great opportunities for the advancement of our understanding of the mechanisms involved in stimulus-response coupling.