Flow Cytometry and Determination of Cell Viability

Pore formation was determined by propidium iodide (PI) staining and subsequent FACS analysis. BY4742 cells were grown in YPD, subsequently shifted in JG medium, and K1 toxin was added to a final concentration of 1,000 AU. Samples were taken over the course of 5 h, washed in 1 × PBS, and fixed in 1% paraformaldehyde (in 1 × PBS). After the transfer to appropriate tubes, 1 μg/ml PI (stock solution 1 mg/ml in PBS, Sigma) was added, and cells were incubated for 10 min on ice. Analysis was performed using BD LSRFortessa Cell Analyzer; data were analyzed with BD FACSDiva^TM software (BD Bioscience, Heidelberg, Germany). Non-stained and heat-treated yeast cells were used for instrument settings; all samples were measured in triplicates with 100,000 gated events each. Samples for determination of cell viability were taken simultaneously, washed with 1 × PBS and plated on YPD agar plates considering appropriate dilutions and replicates.