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 for S100B, a specific protein expressed by these cells. Digitonin-permeabilization of astrocytes has been used to investigate intracellular pools of Ca(2+), 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 microM digitonin permeabilized at least 75% of cells. A range of media with different ionic nature can 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.
Tramontina
F
F
Departamento de Bioquímica, ICBS, Universidade Federal do Rio Grande do Sul, Ramiro Barcelos, 2600-Anexo, 90.035-003, Porto Alegre, Brazil.
Karl
J
J
Gottfried
C
C
Mendez
A
A
Gonçalves
D
D
Portela
L V
LV
Gonçalves
C A
CA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004396:Coloring Agents; D004072:Digitonin / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D009414:Nerve Growth Factors / Q000378:metabolism*; D051381:Rats; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D014343:Trypan Blue
D002135:Calcium-Binding Proteins; D002614:Chelating Agents; D004396:Coloring Agents; D009414:Nerve Growth Factors; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579158:S100b protein, rat; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D014343:Trypan Blue; D004072:Digitonin; D002118:Calcium