Digitonin-permeabilization of astrocytes in culture monitored by trypan blue exclusion and loss of S100B by ELISA

The present protocol details a procedure to permeabilize astrocytes in cultures with digitonin as well as to discuss some data about factors that interfere in permeabilization, particularly divalent cations and nucleotides. Two methods to assess astrocyte permeabilization are described: trypan blue exclusion and ELISA fur S100B, a specific protein expressed by these cells. Digitonin-permeabilization of astrocytes has been used to investigate intracellular pools of Ca2+, internal stores of metabolites, phosphoinositide hydrolysis, and recently we standardized a procedure to study protein phosphorylation (Brain Res, 853 (2000) 32-40). A short incubation time (10 min) with 30 muM digitonin permeabilized at least 75% of cells. A range of media with different ionic nature call be used in cell permeabilization without affecting significantly the extent of permeabilization, but calcium and ATP of the order of 10(-5) M induced a partial resealing which deserves to be considered in assays of permeabilized preparations of astrocytes. (C) 2000 Elsevier Science B.V. All rights reserved.