S3e cells sorter

The fluorescence intensities of M. xanthus strain EM709 simultaneously expressing P_EPS-sfGFP and P_BPS-mCherry were measured by flow cytometry with a Bio-Rad S3E cells sorter. The blue laser (488 nm, 100 mW) was used for the forward scatter (FSC), side scatter (SSC), and excitation of sfGFP, whereas the green laser (561 nm, 100 mW) was used for the excitation of mCherry. Signals were collected using the emission filters FL1 (525/30 nm) and FL3 (615/25 nm) for sfGFP and mCherry, respectively. Cells collected from the colony edges and centers were suspended in TPM and ran at low-pressure mode and at a rate of 10,000 particles per second. The threshold on FSC was 0.12, and the voltages of the photomultipliers were 361, 280, 785, and 862 volts for FSC, SSC, FL1, and FL3, respectively. The density plots obtained (small-angle scattering FSC versus wide angle scattering SSC signals) were gated on the population of interest and filtered to remove multiple events. Populations of 300,000 to 500,000 events were used and analyzed statistically using the FlowJo software. The sfGFP and mCherry signals obtained with the nonfluorescent WT cells were subtracted from the signals obtained with cells of strain EM709. Measurements were carried out 3 times with bacteria from different plates.