Flow cytometry-based analysis of cell integrity after hypoosmotic shock. Wild-type and mutant strains expressing pHluorin-mCherry (SEY6210, LGY83, MTY18 containing pMB517) were grown in the presence of 1 M sorbitol. The cells were either switched to a new medium containing sorbitol (control; 1 M sorbitol > 1 M sorbitol) or to medium without sorbitol (hypoosmotic shock; 1 M sorbitol > 0 M sorbitol) and within ∼1 min analyzed by flow cytometry. (A–C) The plots show the distribution of the pHluorin/mCherry ratio of ∼200,000 cells (readout of cytoplasmic pH). (D) Quantitative analysis of the flow cytometry data collected from wild-type and mutant strains expressing pHluorin/mCherry (MTY93, MTY89, SEY6210, MTY7, MTY2, MTY86, MTY91, MTY11, STY11, LGY83, and MTY18 containing pMB517). “Lysed” cells were defined as cells with a pHluorin/mCherry ratio <1 (indicating loss of PM integrity), whereas “low pH” cells showed a pHluorin/mCherry ratio that was <95% of the control cells but >1 (see panel A). Based on this analysis, the controls of all strains were very similar (shown as “con”). (E) Wild-type and mutant yeast strains expressing pHluorin-mCherry (SEY6210, MTY18, MTY89, STY11, MTY91, MTY86 containing pMB517) were monitored during hypoosmotic shock (1 M sorbitol > 0 M sorbitol) in a microfluidics chamber to determine the percentage of cell lysis (loss of pHluorin signal, indicating loss of PM integrity) and cell rupture (loss of pHluorin and mCherry signal, indicating ruptured cell wall and PM; see Fig. 1 C). The wild type and tcb2/3∆ data are identical to those shown in Fig. 1 C.