Fluorescence activated cell sorting

An overnight culture was diluted 20 times and grown to exponential phase (6 h) to measure the fluorescence profile of the UCC2210, Sir2-tdTomato, tdTomato-Sir2, BY4741, Rad27-tdTomato and Rad52-tdTomato strains by MACSQuant VYB with the MACSQuantify Software (Miltenyi Biotec). A total of 10⁵ cells was analyzed for each strain, and the fsc files were exported and analyzed using the software R (v3.2) with the Bioconductor packages (v3.0). A norm2Filter filter was applied on FSC-A/SSC-A to select homogeneous cells in terms of size, shape, and cellular complexity. tdTomato fluorescence (Channel Y2-A) was log-transformed. All the figures were prepared using the transformed data. Each measurement was repeated three times.

The cell sorting experiments were performed on a MoFlo Astrios EQ cell sorter and analyzed with the Summit v6.3 software (Beckman Coulter). Cells in stationary phase were diluted 100 times and grown at 30° with vigorous shaking (200 rpm) for 16 h prior to cell sorting (final OD ≈ 2). Cultures were spun down at 3000g for five minutes at 4°. Growth media were removed and the cells were re-suspended in ice cold PBS. The SmartSampler and CyClone tube holders were kept at 4° during cell sorting. Cell sorting was carried out with a 70 µm nozzle at an operating pressure of 60 psi. The sorting speed was kept at around 30 000 events per second. The purity mode was chosen for sorting along with a single-drop droplet envelope. Single cells of similar size and granularity were first selected based on the FSC-Area vs. SSC-Area (488 nm laser) plot and the FSC-Height vs. FSC-Area (488 nm laser) plot. Then, based on the tdTomato fluorescence histogram (560 nm laser, 614/20 filter), single cells were simultaneously sorted either into the 2% most and least fluorescent (Figure 2A), or into five subpopulations defined in terms of their fluorescence as follows: 0–20%, 20–40%, 40–60%, 60–80% and 80–100% (only for viability analysis) (Figure 4B).

To analyze the dynamics of recovery of the initial gene expression profile from the sorted extreme subpopulations, the UCC2210:tdTomato-Sir2 strain was grown overnight at 30° in YPD medium and diluted 10 times in the morning. After 3 h, 6.10⁵ cells from the bottom cells and the top 2% were sorted simultaneously with MoFlo Astrios EQ (Beckman Coulter). The unimodality of the sorted cells was verified and they were then grown at 30° in YPD medium. The dynamics of expression recovery was monitored with a MACSQuant VYB flow cytometer (Miltenyi Biotec) for 6 h or 24 h.