Isolation of protoplasmic astrocytes: a procedure based on controlled trypsin digestion.
Trypsinization of rat brain tissue for shorter (10 min) and longer (60-90 min) periods is shown to yield two distinctly different types of dissociated cell populations. The over-all yield of intact dissociated cells declines when the period of trypsinization exceeds 10 min. Comparison of the types of dissociated cells obtained after different lengths of trypsinization indicates that the protoplasmic astrocytes, which represent the bulk of the neuroglial cells in brain, are highly susceptible to degradation during tissue trypsinization. Based on this observation, a new procedure involving controlled trypsin digestion for tissue disruption and Percoll gradient centrifugation has been developed for the isolation of virtually pure populations of intact protoplasmic astrocytes (97-98% particle purity). Identification of the purified cells is based on morphological, histochemical staining, and biochemical (marker enzyme) characteristics.
D000818:Animals; D001253:Astrocytes / Q000166:cytology* / Q000201:enzymology; D001921:Brain / Q000166:cytology* / Q000201:enzymology; D002469:Cell Separation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D005974:Glutamate-Ammonia Ligase / Q000032:analysis; D008858:Microscopy, Phase-Contrast; D051381:Rats; D011919:Rats, Inbred Strains; D014357:Trypsin; D001616:beta-Galactosidase / Q000032:analysis
D001616:beta-Galactosidase; D014357:Trypsin; D005974:Glutamate-Ammonia Ligase