Practical considerations regarding the use of streptolysin-O as a permeabilising agent for cells in the investigation of exocytosis

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Abstract
Streptolysin-O is widely used in cell biological investigations in order to make large (>12 nm) pores in the plasma membrane and so to render the cytosol directly accessible to experimental manipulation. We have compared the effect of streptolysin-O commercially formulated (Murex Diagnostics) as a diagnostic reagent in pathology with two pure reagents (a conventional purified protein, and a recombinant protein generated in E.coli) on exocytotic secretion from mast cells. For mast cells permeabilised by streptolysin obtained from the commercial source, exocytosis (of β-D-N-acetylglucosaminidase) is dependent on provision of both Ca2+ and a guanine nucleotide. In contrast, for cells permeabilised by either of the two pure proteins, a substantial extent of Ca2+-independent exocytosis can be elicited. When the Murex material is subject to dialysis or ultrafiltration, some secretion can be induced in the absence of Ca2+, indicating a modulatory function of the low mol wt additives of formulation, mainly phosphate and cysteine. However, Ca2+-independent exocytosis is still manifest when the pure proteins are reconstituted with ultrafiltrates from the Murex material. These observations indicate that reagents used to permeabilise cells should be characterised thoroughly and used with great care. Confirmation that the cytolytic activity of the Murex material derives from a cholesterol directed factor was demonstrated by inhibition of exocytosis when red blood cell derived (and hence cholesterol containing) sonicated liposomes were provided.