HhAntag691 moa Furthermore, PAbN has been reported to have no inhibitory effect on efflux of ethidium bromide, which is also known as a substrate of ABC-type transporter. What is more, fluorescence of these compounds was not as strong as that of fluorescein, and thus they are not considered to be suitable for the assay in the microfluidic channel. The role of efflux pumps on FDG hydrolysis in E. coli was not fully understood when we chose FDG as a substrate for the assay at the beginning of this study. FDG was defined as a substrate of RND pumps because it was easily hydrolyzed in pump deletion mutants compared with wild-type cells. If FDG was not a substrate of pumps, it will be difficult to discriminate DB from wild-type. Fluorescein was also defined as a substrate of pumps from its accumulation in DC cells. Furthermore, from the results of complete blockage of FDG hydrolysis by protonophore CCCP addition in all of the strains including DC, FDG was defined to be actively imported into the cytoplasm. The lactose permease LacY is not a FDG permease because lacY deletion from DlacI mutants had no effect on FDG hydrolysis in the mutants, and we have yet to identify a FDG permease. Figure 12 shows the proposed AZ 960 mechanism of FDG hydrolysis in E. coli. In wild-type cells, FDG is hardly imported into cytoplasm because FDG is exported by AcrB from periplasm before it is trapped by permease. Rate of FDG influx will increase depending on a concentration of FDG in periplasm until it reach maximum rate. Moderate inhibition of the pumps causes FDG influx and an efflux of fluorescein from the cells by the remaining activity of the pumps, and full inhibition of the pumps results in fluorescein accumulation in cells, similar to what is observed in DC cells. A real pump inhibitor without any effect on bacterial membrane will increase fluorescence in wild-type cell by concentration dependent manner and will cause increased accumulation of fluorescence in the cells. In fact, we detected moderately increasing fluorescence and increased accumulation of fluorescence in DBC/pABM according to increasing concentration of D13-9001. In contrast, peameabilization of outer membrane will cause leakage of fluorescein from DC cells, and peameabilization of inner membrane will efficiently increase FDG influx and fluorescein production to release it from cells with or without pumps. By the FDG assay, it is easy to detect outer membrane peameabilization by disappearance of fluorescein accumulation in DC cells, and inner membrane peameabilization by increased fluorescence especially in the medium of pump deletion mutants. Determination of efflux pump inhibition activity via the typical methods of measuring the influx or efflux of some substrates by their fluorescence with a plate reader makes it difficult to exclude the effect of outer membrane peameabilizing activity. In addition, accurate estimation of FDG hydrolysis through monitoring fluorescence with a plate-reader is impractical because the total fluorescence of fluorescein is higher when diffused in the medium than when accumulated in the cells, and fluorescence determined by a plate reader was usually higher in DB than in DC. Therefore, fluorescence determined by a plate reader does not accurately correlate with the amount of fluorescein produced, and it is difficult to estimate the inhibitory effect on pumps with FDG by a plate reader. The microfluidic channel method enables discrimination of pure efflux pump inhibition from membrane permeabilization.