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.