Lectin staining and flow cytometry

Glass culture tubes were inoculated with 1 mL of P. aeruginosa in LB or Jensen’s minimal media at an OD₆₀₀ 0.8 and incubated statically at 37°C for 4 hr. Non-adhered cells were removed by washing three times with 2 mL sterile phosphate buffered saline (PBS). Biofilm cells were harvested by vortexing in 1 mL PBS with tetramethylrhodamine (TRITC) conjugated lectins (TRITC-labeled WFL lectin (100 μg/mL; Vector Laboratories) for Pel, TRITC-labeled HHA (100 μg/mL; EY Laboratories) for Psl) and incubated on ice for 5 min. Cells were washed 3 times to remove non-adhered lectin, resuspended in PBS, and immediately analyzed for GFP and TRITC fluorescence on a BD LSRII flow cytometer (BD Biosciences). Events were gated based on forward and side scatter to remove particles smaller than a single P. aeruginosa cell and large aggregates.

We used PAO1 cells that did not express GFP (wild type PAO1; Figure 1—source data 2) or constitutively expressed GFP (PAO1 Tn7::P(A1/04/03)::GFPmut; Figure 1—source data 2) to define a gate for high GFP fluorescence. We validated this gate using a strain in which we expect very high levels of reporter activity (surface grown PAO1 ΔwspFΔpelAΔpslBCD harboring pPcdrA::gfp_ASV) and saw that 91.6% of cells had high GFP levels (Figure 1—source data 2), in agreement with our flow cell characterization of this strain (Figure 2A). We determined gating for TRITC using cells that had not been stained with TRITC-conjugated lectin (Figure 1—figure supplement 6A), as well as two strains that overproduced either Psl (Figure 1—figure supplement 6B) or Pel (Figure 1—figure supplement 6C) that were stained with the appropriate TRITC-conjugated lectin. Our flow cytometry gating procedure accurately gated 99.7% of wild type PAO1 cells (without the PcdrA reporter or lectin-staining) as low GFP and low TRITC (Figure 1—figure supplement 6D).