Quantification of Fluorescent Neomycin Uptake

Quantification of fluorescent neomycin (Neo-cy5) uptake was performed as described [61 (link)]. Neo-cy5 is an aminoglycoside coupled to the fluorophore Cy5, and has been shown to be active against Gram- bacteria [27 (link)]. Briefly, overnight cultures were diluted 100-fold in rich MOPS (Teknova EZ rich defined medium). When the bacterial cultures reached an OD₆₀₀ of 0.25, they were incubated with 0.4 μM of Cy5 labeled Neomycin for 15 minutes at 37°C. 10 μL of the incubated culture were then used for flow cytometry, diluting them in 250 μL of PBS before reading fluorescence. Flow cytometry experiments were performed as described [62 (link)]. For each experiment, 100000 events were counted on the Miltenyi MACSquant device.