Toxicity of agar for neutrophilic colonies

The soft agar culture system is widely used to study in vitro regulation of granulopoiesis. This report is presented to illustrate how agar itself affects the kinetics of colony growth and thus, the results obtained with the assay. The main finding was that agar caused cells to lyse as they matured. Death appeared to occur at the myelocyte/metamyelocyte transition. Thus colonies did not develop a non-growth fraction of metamyelocyte, bands and segmented forms. Colonies grew in a multiphasic fashion owing to the maturation/differentiation process. Growth was characterized by an initial logarithmic phase, followed by a phase of non-logarithmic growth in which the size of colonies depended on the relative rates of cell production and loss. When clonogenic cells exhausted their self-renewal capacity, colonies entered a decay phase, during which they decreased in size and disappeared. In pulsing experiments the onset of decay phase was delayed if the addition of growth factor was delayed. Conversely, the onset of the decay phase was premature if mitosis was arrested with vincristine. During the decay phase the proportion of cells with damaged membranes (permeable to propidium iodide) remained constant. It was concluded that data accumulated during the decay phase cannot be interpreted reliably. This point should be considered in designing experiments and interpreting the results with this assay system.