Synergy mx 96 well plate reader

The Resazurin assay (REMA) was performed in order to evaluate the impact of the compounds on the respiratory activity after a short period of time [23 (link),36 (link)]. Cells of B. subtilis or X. citri were incubated in contact with resazurin in the same medium used for the MIC evaluations (DMM and Xam1 respectively) in 96-well plates. The compound BC1 was used at a final concentration of 40 µg/mL for B. subtilis and 30 µg/mL for X. citri; T9A was used at 40 µg/mL for B. subtilis and 20 µg/mL for X. citri. Rifampicin 0.625 µg/mL, ciprofloxacin 0.625 μg/mL, tetracycline 10 μg/mL, and vancomycin 1.0 μg/mL were used as controls for B. subtilis, and rifampicin 0.625 µg/mL, ciprofloxacin 10.0 μg/mL, tetracycline 10 μg/mL, and penicillin G 800 μg/mL were used as controls for X. citri, along with samples without added compound. Resazurin was added to a final concentration of 0.1 mg/mL. Fluorescence (excitation 530 nm, emission 560 nm, bandwidth 9 nm) was recorded every 20 min up to 120 min in a BioTek Synergy Mx 96-well plate reader (BioTek instruments, Winooski, VT, USA).

Nonfluorescent resazurin can be reduced to fluorescent resorufin in a NAD(P)H dependent manner in the presence of NAD(P)H dehydrogenase (De Jong and Woodlief, 1977; Barnes and Spenney, 1980; Winartasaputra et al., 1980; Hanson and Freier, 1983; Shahangian et al., 1984). Therefore, the resazurin/NAD(P)H dehydrogenase/NAD(P)H system can be used to detect the NAD(P)H level of cells. X. campestris pv. campestris cells were grown in LB broth until OD₆₀₀ reached 0.1. Resazurin was added to the culture at a final concentration of 0.1 mg ml⁻¹. Relacidine B, rifampicin, and polymyxin B were added at final concentrations of 0.25, 0.125, and 1 μg ml⁻¹ respectively. DMSO was used as a negative control. Blanks (without cells) revealed that the signal change is not caused by the interaction of resazurin and compounds. Fluorescence was recorded at a wavelength of 560 nm every 20 min over a period of 4 h with a BioTek Synergy Mx 96‐well plate reader.

The ATPase activity of ECF–FolT2 reconstituted in proteoliposomes (loaded with 0 or 100 nM of folate as described above) was measured by using a coupled enzyme assay, in which the amount of ADP produced (and thus the amount of ATP hydrolysed) is coupled stoichiometric with the oxidation of NADH42 (link). The assay was performed at 30 °C in a 96-well plate and the absorbance at 340 nm was measured by a Synergy MX-96-well plate reader (BioTek Instruments, Inc.). A volume of 200 μl of reaction solution per well contained 50 mM KPi, pH 7.5, 200 mM NaCl, 5.2 nM (1.2 μg) of ECF–FolT2, 4 mM sodium phosphoenolpyruvate, 0.3 mM NADH and 3.5 μl of pyruvate kinase/lactic dehydrogenase enzyme mixture from rabbit muscle (Sigma-Aldrich) in 50% glycerol. The reaction solutions were supplemented with 0 or 100 nM folate as indicated (folate was thus present both on the inside and the outside of the proteoliposomes in the 100 nM folate condition). After incubation of the reaction solutions for 3 min at 30 °C, 1 mM of MgATP, pH 7.5, was added to each of the reactions and the absorbance of NADH at 340 nm was followed for 7 min. The ATPase activity was expressed in μmol of ATP hydrolysed per min per mg of ECF–FolT2).