Tramontina F, Karl J, Gottfried C, Mendez A, Gonçalves D, Portela L V, Gonçalves C A
Departamento de Bioquímica, ICBS, Universidade Federal do Rio Grande do Sul, Ramiro Barcelos, 2600-Anexo, 90.035-003, Porto Alegre, Brazil.
Brain Res Brain Res Protoc. 2000 Nov;6(1-2):86-90. doi: 10.1016/s1385-299x(00)00041-6.
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.
本实验方案详细介绍了一种用洋地黄皂苷使培养的星形胶质细胞通透化的方法，并讨论了一些关于影响通透化的因素的数据，特别是二价阳离子和核苷酸。文中描述了两种评估星形胶质细胞通透化的方法：台盼蓝排斥法和针对这些细胞表达的特异性蛋白S100B的酶联免疫吸附测定（ELISA）法。星形胶质细胞的洋地黄皂苷通透化已被用于研究细胞内的钙离子池、代谢物的内部储存、磷酸肌醇水解，最近我们还标准化了一种研究蛋白质磷酸化的方法（《脑研究》853 (2000) 32 - 40）。用30微摩尔/升洋地黄皂苷短时间孵育（10分钟）可使至少75%的细胞通透化。一系列具有不同离子性质的培养基可用于细胞通透化，而不会显著影响通透化程度，但10^(-5) M量级的钙和ATP会导致部分重新封闭，这在星形胶质细胞通透化制剂的检测中值得考虑。
