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Summary in 96-Well Microplate-Based Cytotoxicity and Development of High-Content Image Analysis (HCIA) Assay with High Throughput and High Accuracy

Toshihiko Kasahara

Various cytotoxicity assays using 96-well microplate have often been performed by measuring biological indicators, such enzyme activity, uptake quantity of dye, or cellular ATP content, and as a matter of course, they have been reported in several manuscripts as a tool for expressing cell viability. However, recently reported that cytotoxicity assays, such as ATP and MTS, were underestimated cytotoxicity when chemicals such anti-cancer drug or mutagens induced cell hypertrophy with increasing intracellular ATP content. In addition, there are few studies using many chemicals to compare the cell viability of the cytotoxicity test using the abovementioned indicators and the actual cell counting. Therefore, the authors have revealed that cytotoxicity tests using biological indicators, such as those mentioned above, do not necessarily produce cell viability that accurately reflects the number of cells as target for 25 chemicals. Moreover, authors clarified that cell hypertrophy and cell cycle are correlated with the use of HCIA (high-content image analysis). Based on these results and the authors’ experience, we summarized the features of the 96-well microplate-based cytotoxicity test. Finally, the authors demonstrated the usefulness of the HCIA assay, which can obtain a large number of cytological parameters at once from a single microplate by using cell cycle-specific inhibitors.