2.2 Flow cytometry and fluorescence-activated cell sorting (FACS)
Flow cytometry analysis and cell sorting (FACS) was done on a BD FACSAria IIIu (BD Biosciences) equipped with BD FACSDiva software v.6.1.3 (BD Biosciences) for general operation. GFP and SYBR Green were excited by a 488 nm laser (20 mW) and detected on a 530/30 nm bandpass filter. The mCherry signal was excited by a 561 nm laser (50 mW) and detected on a 610/20 nm bandpass filter. Fluorescent minus one (FMO) controls of mCherry, GFP and non-fluorescent samples were used to set PMT voltages and appropriate gating. Samples were diluted in a 0.9% w/v NaCl solution until reaching ~3000 evt/s, except for counting, where they were diluted 200X. Events were recorded for 60 sec at a flow rate of ~15 µl/min. Thresholds on the forward scatter (FSC) were kept at 1200 with a “AND” threshold on the side scatter (SSC) kept at the minimum 200. The gating strategy can be seen in supplementary figure 3. Transconjugant cells were identified by sorting 10500 gfp expressing cells with purity precision and sample purity was subsequently analyzed by recording the sorted sample for 60 seconds.