The isolation of cerebral neurons with partial retention of processes.
A novel tissue disaggregation technique has been devised which permits the isolation of neurons with fairly extensive processes attached. Cortex is dissociated by aspiration through nozzles of decreasing size followed by agitation on a vortex mixer, rather than by the usual technique of forcing tissue through sieves. After each aspiration step, dissociated cells are separated from undisrupted tissue by coarse filtration and the latter is subjected to repeated treatment. This prevents unnecessary trauma to the free cells. After disruption is complete, small pieces of undisrupted tissue are removed from the cell suspension by floating on the foam created by degassing the suspension under vacuum. Cells are purified by conventional velocity-gradient centrifugation. This procedure has been applied successfully to fresh rat brain, with or without a preincubation with trypsin, frozen human brain and frozen bovine brain. The cell yields from rat brain were comparable to or better than, those obtained by other procedures (37 X 10(6) cells/g brain) while the purity was comparable. Cell yields from human brain were similar to those from rat brain but the purity was lower. The lowered particle purity of human and bovine cells can probably be attributed to the conditions of storage of the tissue and to trapping of free nuclei in the meshwork of dendritic processes. Values are given for the amount of protein, RNA and DNA per cell.
D000818:Animals; D001253:Astrocytes / Q000166:cytology; D002417:Cattle; D002469:Cell Separation / Q000379:methods*; D002540:Cerebral Cortex / Q000166:cytology*; D004247:DNA / Q000032:analysis; D006801:Humans; D009474:Neurons / Q000032:analysis / Q000166:cytology*; D051381:Rats
D004247:DNA