Aminoglycoside Uptake Assay Protocol

The aminoglycoside uptake assay was done according to Loughman et al. (2016) (link). GEN was tagged using the Texas Red-X succinimidyl ester (ThermoFisher) at a molar ratio of 300:1 and incubated overnight at 4°C with agitation. To remove unbound Texas Red, the dye remover column (Thermo Fisher) was used according to the manufacturer’s protocol. Bacteria were adjusted to an A_600nm of 0.1 and incubated 2 h at 35°C with agitation in the presence of 128 μg/ml of the Texas Red-GEN conjugate. When TO was added during the uptake assay, it was used at 8 μg/ml. Plates were read with a TECAN Genios Fluorescence plate reader with the 535/595 nm filters.