FIGURE 4. A fluorescent reporter for CN activity allows for simultaneous exclusion of dead cells via flow cytometry.(A) Representative micrographs of wild type and Δpmr1 cells equipped with pAMS366-4xCDRE-GFP^PEST and stained with propidium iodide (PI) to indicate loss of plasma membrane integrity and thus cell death. Scale bar = 5 µm. (B-G) Flow cytometric analysis of PI-stained aged wild type cells treated with Ca²⁺ for 1 h as well as Δpmr1 cells equipped with pAMS366-4xCDRE-GFP^PEST. Respective dot plots (B, E), histograms of GFP^PEST intensities of PI negative (live) and PI positive (dead) cells (C, F) and mean GFP^PEST intensities of total, live and dead cell populations (D, G) are shown. (H) Simultaneous quantification of cell death via PI staining and CN activity in cells equipped with pAMS366-4xCDRE-GFP^PEST. Wild type and Δpmr1 cells of different age and upon treatment with different concentrations of Ca²⁺ were analyzed, and the difference of GFP^PEST fluorescence intensity between total and live cell populations (ΔGFP^PEST (live-total)) was plotted against the percentage of PI positive cells.