Easycyte ht flow cytometer

Co-cultures were generated by mixing equal proportions (1 OD₆₀₀) of cells expressing fluorescent GFP and non-fluorescent GFP-Y66F in 10 mL of synthetic complete media. Immediately following mixing, approximately 0.15 optical densities were collected, washed, resuspended in 2 mL of sodium citrate buffer (50 mM sodium citrate, 0.02% NaN₃, pH 7.4), and stored at 4°C for subsequent flow cytometric analysis. All co-cultures were then diluted to 0.004–0.01 optical densities in 10 mL of synthetic complete media with or without stress (0.2 M CaCl₂ or 0.4 M KCl) and incubated at 30°C with continuous rolling. Where indicated, media was supplemented with 250 μM reduced L-glutathione (Sigma), 250 μM DL-homocysteine (Sigma), or 5 mM L-ascorbic acid (Sigma). Co-cultures were sampled and diluted to 0.004–0.02 optical densities, as described above, every 12 hours to ensure that the population did not exceed logarithmic growth during the duration of the experiment. Following sonication, the percentage of GFP and GFP (Y66F) at each timepoint was quantified using a Guava EasyCyte HT flow cytometer and analyzed with FlowJo software. Replicates in which a fitness difference was observed between the GFP and GFP (Y66F) controls were excluded from the final analysis.