Facsuite cs t research beads 650621

Strains were cultured at 30°C and 240 RPM for approximately 18 h—in order to reach the steady state—in 2 ml SDC (supplemented with 2% glucose or galactose) or SDC supplied with different concentrations of copper/methionine. 20 μl of cell solution were then diluted into 300 μl ddH2O before any FACS measurement. We measured the fluorescence intensity from our strains (due to yEGFP) with a BD FACSVerse machine (laser 488 nm-FITC filter 527/32 nm). In order to have reliable measurements the BD FACSVerse should have passed the Performance Quality Control (PQC). Moreover, each PQC was followed by fluorescent beads (BD FACSuite CS&T Research beads 650621) measurement to adjust the FITC voltage. If the relative difference between the values of the bead peak in two independent experiments was not bigger than 5%, then the machine conditions had not changed considerably during these experiments and the collected data was reliable (see the diagram in Supplementary Figure SMM1). All FACS files were analyzed via the flowcore R-Bioconductor package (37 (link)). Ten thousand cells were collected during each measurement. Fluorescence mean values were calculated on at least three independent experiments. We used two-sided Welch's t test to do the statistical analysis (all fluorescent data collected via FACS is available in Supplementary Material Excel sheet 3).

Strains were cultured, first, at 30°C 240 RPM in 2 ml SDC or SDC supplied with methionine (10 mM), GA3 or ABA for 16 h. Then, cell solutions were diluted (1:20) into the same medium to grow for 8 more hours. 20 μl cell culture were then added to 300 μl ddH2O. BD FACSVerse machine was used for fluorescence intensity evaluation (laser 488 nm-FITC filter 527/32 nm, and laser 405 nm-Pacific Blue filter 448/45 nm were selected for green and blue fluorescence detection, respectively). To gain reliable data, we checked the performance quality control (PQC) of the machine and used fluorescent beads (BD FACSuite CS&T Research beads 650621) to regulate the voltage with FITC and Pacific Blue filter. The criterion to adjust the voltage is that the difference of the peak values between two consecutive bead measurements should not be bigger than 5%. Ten thousand cells were collected in each independent experiment (three at least) and fluorescence mean value—analyzed with the flowcore R-Bioconductor package (27 )—was utilized for data analysis and comparison. Statistical analyses were based on two-sided Welch's t test and one-way ANOVA (for all FACS analyses, see Supplementary Tables S1–S23 and S26–S27 in the section ‘Supplementary Tables with Statistical Data Analysis’ and Supplementary Material Excel sheet 3).