CELL-DENSITY DEPENDENT PLATING EFFICIENCY AFFECTS OUTCOME AND INTERPRETATION OF COLONY-FORMING ASSAYS

Background and purpose. The usefulness of colony forming assays (CFA) has been established for almost 40 years (Puck and Marcus, J. Exp. Med . 103: 653-666, 1956). Although time-consuming and not successful for all cell lines, it is generally considered to be the gold standard of assays for testing the sensitivity of cell lines to ionizing radiation or other cytotoxic agents in vitro. We recently found for several cel l. lines that the plating efficiencies of both control and irradiated cells is dependent upon the density of cells seeded for colony formati on; that is, increasing cell inoculum levels resulted in a non-linear relationship with colony formation, even at relatively low colony numb ers. Material and methods. All data from a human melanoma cell line, t ransfected with c-myc or N-ras, as well as from normal human diploid f ibroblasts, were taken to see how this phenomenon influenced outcome a nd interpretation of clonogenic assays. Survival was recalculated usin g all data, or only data with a linear relationship between inoculum l evel and colony formation. Results. It is found that when data with a non-linear relationship between inoculum level and colony formation ar e included, survival can be underestimated due to inhibition of colony formation in treated cultures. Conclusion. For validity, colony formi ng assays must be standardized to assure a constant relationship betwe en the cell density and colony forming efficiency, This usually requir es a much lower density of colonies than has been typically published for many cell survival studies.