In vitro reaggregation of dissociated mouse cerebellar cells: I. Demonstration of different aggregation mechanisms

Abstract

Reaggregation of mechanically dissociated mouse cerebellar cells (M cells) was compared with cells that received an additional trypsinization either before (T cells) or after (MT cells) the dissociation step. Reaggregation behaviour was followed by measuring the number and size distribution of particles with a Coulter counter. Aggregation rates which were calculated as percentage of decrease of particles could be measured reproducibly. Since the percentage of very large particles (> 100 cells) formed during aggregation varied considerably from one experiment to the next, size distribution curves of particles were used more to distinguish qualitative differences in a less quantitative way. Whereas aggregation rates and size distribution of particles with M cells were almost identical when aggregation occurred in medium of high (1.1 mM) or low (0.1 mM) Ca 2+ concentrations, T and MT cells aggregated better at high Ca 2+ concentration. Their aggregation rates were reduced by approx. 50% at low Ca 2+ concentrations and larger aggregates were hardly formed under these conditions. The aggregation rates of T and MT cells showed a clear dependence on Ca 2+ concentration, being half maximal at approx. 0.1 mM Ca 2+. The ability of M cells to aggregate at low or high Ca 2+ concentrations was influenced by subsequent trypsinization to produce MT cells. When the trypsin concentration was changed from 0.001 to 0.1% during this procedure the aggregation rates at high Ca 2+ concentration were reduced to approx. 80% of the maximal value, whereas those at low Ca 2+ concentrations were reduced to 35%. Variation of the Ca 2+ concentration between 1.1 and 0.1 mM during the trypsinization step (0.015% trypsin) revealed no difference on the aggregation rates. We propose that M cells aggregate mainly or exclusively by a Ca 2+-independent binding mechanism, whereas T or MT cells aggregate using a Ca 2+-dependent one which may be functionally silent in M cells.