Effects of chemical exposure on MMP was assessed using rhodamine123 (rho123) (sigma Aldrich, R8004) using the protocol described previously (van der Stel et al. 2020 (link)). Cells were seeded two days before exposure. At the day of exposure, cells were first co-stained with 200 ng/µl Hoechst (Life technologies, H1399) and 1 µM rho123 for 75 min, followed by a co-staining of 0.2 µM rho123 and 100 nM propidium iodide (PI) (Sigma-Aldrich, P4170) and exposure to the desired concentration of test chemical. Over a period of 24 h, the signal intensity of Hoechst (408 nm), rho123 (448 nm), and PI (561 nm) were monitored every hour. The nuclei were identified based on the Hoechst images and formed the basis for the assessment of the cytoplasm (defined as those pixels within a maximal distance of 10 pixels around the nucleus). Finally, the intensity of the rho123 was assessed in the cytoplasm, and cell death estimates were based on the fraction of nuclei that showed at least 10% of the pixels also positive for PI-staining. Part of the MMP data were used for development of a dynamic mathematical model and integration with RNA-seq data elsewhere (Yang et al. 2021 (link), van der Stel et al. 2021 (link)).
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