Spillover inverts a ratio-normalised viability readout in yeast flow cytometry - BG10RFP

Raw flow cytometry data (FCS 3.0) of Komagataella phaffii BG10-RFP, an isogenic derivative of BG10 with a constitutively expressed red fluorescent protein, for the article “Spillover inverts a ratio-normalised viability readout in yeast flow cytometry”. Heat-treated (65 °C, 15 min) and untreated cells were mixed at nominal ratios of 0, 25, 50, 75 and 100 % living cells, stained with 5(6)-CFDA in intracellular pH buffer at pH 3.5, and measured in three independent replicates, each prepared from its own culture. The dataset contains 18 files: 15 stained mixtures, two unstained controls and one unstained control from a separate acquisition. File names give the nominal percentage of living cells and the replicate (culture), e.g. 25_2; Unstained_100 is the untreated and Unstained_RFP_0 the heat-treated unstained control. Unstained_BG10RFP_I_1 holds unstained, untreated BG10-RFP in the same buffer from a separate acquisition, used to replicate the spillover measurement against BG10. Acquisition stopped after 20 000 events within a gate on red fluorescence set on the parental strain; because the red fluorescent protein shifts living cells out of this gate, files of living-rich tubes contain more events. All data were acquired on one CytoFLEX LX (Beckman Coulter; five lasers, nineteen fluorescence detectors). Every file is the complete, ungated acquisition of one tube. CFDA is recorded in FITC-A (488 nm excitation, 525/40 nm), forward scatter as FSC-A.