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Cell proliferation assays are widely applied in biological sciences to understand the effect of drugs over time. However, current methods often assess cell population growth indirectly, that is, the cells are not actually counted. Instead other parameters, for example, the amount of protein, are determined. These methods often also demand phototoxic labels, have low temporal resolution, or employ end-point assays, and frequently are labor intensive. We have developed a robust and label-free kinetic cell proliferation assay with high temporal resolution for adherent cells using digital holographic microscopy (DHM), one of many quantitative phase microscopy techniques. As no labels or stains are required, and only very low intensity illumination is necessary, the technique allows for noninvasive continuous cell counting. Only two image processing settings were adjusted between cell lines, making the assay practical, user friendly, and free of user bias. The developed direct assay was validated by analyzing cell cultures treated with various concentrations of the anti-cancer drug etoposide, a well-established topoisomerase inhibitor that causes DNA damage and leads to programmed cell death. After treatment, the unstained adherent cells were nondestructively imaged every 30 min for 36 h inside a cell incubator. In the recorded time-lapse image sequences, individual cells were automatically identified to provide detailed growth curves and growth rate data of cell number, confluence, and average cell volume. Our results demonstrate how these parameters facilitate a deeper understanding of cell processes than what is achievable with current single-parameter and end-point methods. © 2017 International Society for Advancement of Cytometry.

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