Neomycin Binding Assay via Flow Cytometry

Experiments were performed as described ^{60 (link)}. Briefly, overnight cultures were diluted 100 fold MOPS Rich medium. When the bacterial strains reached an OD of 0.25, cells were incubated them with 0.4 μM of Cy5 labeled Neomycin for 15’at 37°C. Then, 20 μl of the incubated culture were used for flow cytometry, diluting them in 200 μl of PBS before reading. Flow cytometry experiments were performed as previously described ^{61 (link)}. For each experiment, 50 000 events were counted on the Miltenyi MACSquant device. The Y3 fluorescence channel was then used to measure fluorescence intensity.