CeDaD-a novel assay for simultaneous tracking of cell death and division in a single population

Abstract

The cell division cycle and the various forms of programmed cell death are interconnected. A prominent example is the tumor suppressor p53, which not only induces apoptosis but also plays an important role in the arrest of the cell cycle. Consequently, simultaneous analysis of cell division and cell death is frequently of significant interest in cell biology research. Traditionally, these processes require distinct assays, making concurrent analysis challenging. To address this, we present a novel combined assay, called CeDaD assay-Cell Death and Division assay-which allows for the simultaneous quantification of cell division and cell death within a single-cell population. This assay utilizes a straightforward flow cytometric approach, combining a staining based on carboxyfluorescein succinimidyl ester (CFSE) to monitor cell division with an annexin V-derived staining to assess the extent of cell death.

Fig. 1. Cell count and WST assay indicate an impact of three compounds on cell population growth, but cannot differentiate between cell cycle arrest and cell death induction.

HCT116 wt cells were treated with varying concentrations of AMG 232 (A), YKL-5-124 (B), or volasertib (C) for 2 days. Relative absorbance (A₄₅₀) in WST assay and relative cell count by cell counting compared in relation to the DMSO control (n ≥3). The concentration-dependent effect of volasertib on cell count was compared after 2, 3, and 4 days of volasertib treatment (D, n ≥3). Cell counting 2 days after treatment with drug concentrations that were used for further tests was compared between treatments (E, n ≥3). Mean ± SD are given, and P values were calculated by two-way ANOVA (*P <0.05; **P <0.01; ***P <0.001). Concentration-response curves were calculated by least square regression with variable hill slope.

Fig. 2. Cell division and cell death rates can be analyzed from one cell sample.

HCT116 wt cells were treated for 2 days with AMG 232, YKL-5-124, or volasertib. Representative CellTrace Violet staining of DMSO control and 10⁻⁶ M volasertib-treated cells was gated by number of cell divisions within 2 days (A). Cell division distribution was compared between both conditions (B, n = 3). Based on the cell division analysis and following the appropriate equation (C) cell division rates were calculated as average cell divisions per day (D). For cell death analysis, cell populations were gated by Apotracker Green and propidium iodide (PI) staining. In representative gating analyses of DMSO control (E) and 10⁻⁶ M Volasertib-treated cells (F) were gated as viable, apoptotic, dead, or artifact. Cell death rates represented by apoptotic and dead cell populations were compared between treatments (G, n = 3). Cell division rates (H) and cell death rates (I) of the DMSO control and after volasertib treatment (10⁻⁶ M and 10^−7.5 M) were compared between HCT116 wt and LIN37^−/−/RB^−/− cells. Mean ± SD are given, and P values were calculated by two-way ANOVA (*P <0.05; **P <0.01; ***P <0.001).

Fig. 3. Cell division and cell death rates adequately predict cell growth behavior measured by cell count.

Cell division and cell death rates calculated for HCT116 wt cells treated with AMG 232, YKL-5-124, or volasertib for 2 days were used to calculate a growth prognosis using the appropriate exponential growth equation (A). Calculated fold changes of cell number after 2 days of treatment were compared with each other (B, n = 3) and with cell count data (C, n ≥3). The growth behavior can also be described as a process by the exponential growth curve and the calculated fold changes of the cell number per day (D, n = 3). The calculated cell growth after treatment with 10^–6 M volasertib was compared with cell count over 4 days after treatment (E, n ≥3). Exponential growth curves of the CeDaD assay data (blue line) as well as the cell counting data from day 0 to 4 (yellow line), the timeframe of the CeDaD assay (day 0 to day 2, dark-green line), and the timeframe after CeDaD measurement (day 2 to day 4, pink line) were generated. All exponential growth curves were calculated by least square regression and plotted with 95% confidence interval. Mean ± SD are given, and P values were calculated by two-way ANOVA (*P <0.05; **P <0.01; ***P <0.001).