Comparison of the cell kill measured by the Hoechst-propidium iodide flow cytometric assay and the colony formation assay.

Abstract

A flow cytometric live-dead cell assay that uses the dual staining of Hoechst 33342 and propidium iodide (HO-PI) was evaluated for its ability to determine the clonogenicity of treated HeLa cells. The colony-forming assay was used as the reference to determine the capability of the HO-PI assay to measure the proportion of clonogenic cells present in a given population. The viability estimates of both the FCM and trypan blue dye exclusion assays accurately predicted the colony-forming efficiency (CFE) of untreated populations of HeLa cells. However, immediately after treatment with either heat, freeze-thawing, or ionizing radiation the HO-PI assay greatly overestimated the clonogenicity of HeLa cells. This lack of correlation between the FCM determined viability and clonogenic survival was also observed when the cells were heated (45 degrees C, 30 min) and then assayed at 0, 1, 2, 3, 6, 12, 16 or 23 hr after treatment. These data demonstrate that viability as estimated by the HO-PI did not predict survival after acute treatment. In sixty-seven mouse mammary tumour cells, when clonogenicity decreased as a function of time spent in nutrient deprived conditions (chronic treatment), the HO-PI assay again predicted higher CFEs than were measured. Therefore, in these experiments the HO-PI assay could not predict cell death and gave no better measure of cell viability than the trypan blue dye exclusion test.