Flow cytometry data – W34/70 (S23): dual-parameter replicative age and viability profiling of Saccharomyces pastorianus

W34/70 (Experiment ID: S23) — Saccharomyces pastorianus Strain: SafLager™ W-34/70 (Saccharomyces pastorianus), industrial lager yeast. Role: method development. Data: 96 FCS files from a flow cytometry experiment. Cells were measured in two buffer systems at four pH levels each: ICP buffer (pH 3, 4, 5, 6) and MOPS buffer (pH 4, 5, 6, 7), with three biological replicates per condition. Measurement conditions include unstained controls (R, RI), CFDA-stained samples (CFDA, CFDAI), and mCherry-expressing cells with and without CFDA staining (Age_I, Age_M, Age_I_CFDA, Age_M_CFDA). Analysis: FCS files were processed using a custom Python pipeline. The workflow comprises: (1) rectangular pre-gating on FSC-H × PC7-H to remove debris and aggregates; (2) Mahalanobis distance filtering (99.9th percentile, χ², df = 2) for 2D outlier removal; (3) subsampling to 20,000 events per file; (4) viability classification via the FITC-A/PerCP-A ratio using CFDA fluorescence (threshold 0.7 on log₁₀ scale, scale group A); (5) Gaussian Mixture Model (GMM) fitting on the ECD-A channel (mCherry) to separate replicative age subpopulations (superpeak approach, 3–5 components). Extended pre-gate boundaries were applied for MOPS pH 4 and 5 to compensate for pH-induced elevated PC7 autofluorescence. Viability thresholds and GMM parameters were developed and validated using this strain.