Quantitative electronic analysis of normal and transformed BHK21 fibroblast aggregation.

The kinetics of cell aggregation in shaking suspension are complex. Consequently most existing forms of assessment are qualitative or semiquantitative. We have used a Coulter counter coupled to a particle size discriminator to describe aggregation more precisely. BHK21 C13 cells were suspended by light or heavy trypsin/EDTA treatments, and then aggregated at 37 °C. Plots were obtained showing the distribution of cells in 1-50 cell aggregates, from which were calculated the rates of redistribution of cells between aggregates. About half the cells in the initial suspension were not single. During the first 5 min of aggregation single cells and aggregates containing up to 4 cells disappeared into larger aggregates. At later times there was a shift towards a net loss of slightly larger aggregates. Adhesions between single cells were the most common event throughout aggregation, but they declined relatively rapidly with time as adhesions between aggregates became relatively more prominent. The overall rate of adhesions declined 10-fold within 12 min and 100-fold within 90 min. In contrast to normal fibroblasts, nearly all transformed BHK21 cells in initial suspensions were single, and even by 30 min few single cells had adhered to form aggregates. The initial rate at which adhesions were formed was only about 15 % of that of normal cells. Heavy trypsin/EDTA treatment of normal cells released DNAse-sensitive material which markedly altered the aggregation kinetics. These changes included the disaggregation of small loosely adhering cell clusters coupled with the formation of abnormally large aggregates. It is suggested that careful preparation of suspensions together with assessment of aggregation on the basis of initial adhesion rate will improve the accuracy and information yield in this type of experiment.