Quantifying Fluorescent Neomycin Uptake in Bacteria

Quantification of fluorescent neomycin uptake was performed as described (29 (link)). Neo-Cy5 is an aminoglycoside coupled to the fluorophore Cy5 and has been shown to be active against Gram-negative bacteria (31 (link), 55 ). Briefly, overnight cultures were diluted 100-fold in rich MOPS (Teknova EZ-rich defined medium). When the bacterial strains reached an OD 600 nm of ∼0.25, they were incubated with 0.4 µM of Cy5-labeled neomycin for 15 min at 37°C. Ten microliters of the incubated culture was then used for flow cytometry, diluting them in 250 µL of PBS before reading fluorescence. WT V. cholerae was incubated simultaneously without Neo-Cy5 as a negative control. Flow cytometry experiments were performed as described (56 (link)) and repeated at least three times. For each experiment, 100,000 events were counted on the Miltenyi MACSquant device.