Intracellular ATP levels of E. coli B2 were determined using an Enhanced ATP Assay Kit (Beyotime, China). E. coli B2 suspension was incubated with thymine (0, 5, and 10 mM) for 1 h. After incubation, bacterial cells were harvested and lysed with lysozyme, and the supernatant was prepared for ATP levels measurement. Detecting solution was added to a 96-well plate and incubated at room temperature for 5 min. Subsequently, the supernatants were added to the wells and its luminescence was measured by Infinite M200 Microplate reader (Tecan). Total ATP levels were calculated from the luminescence signals accordingly.
2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM) were added into E. coli B2 suspension (Liu et al., 2020a (link)). After incubated at 37°C for 30 min, 190 μL of probe-labeled bacterial cells were added to a 96-well plate and 10 μL of ciprofloxacin (0, 1, 5, and 10-fold MIC) without or with thymine (10 mM) were added. After incubation at 37°C for 1 h, fluorescence units were immediately measured with the excitation wavelength at 488 nm and emission wavelength at 525 nm using an Infinite M200 Microplate reader (Tecan).
2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM) were added into E. coli B2 suspension (Liu et al., 2020a (link)). After incubated at 37°C for 30 min, 190 μL of probe-labeled bacterial cells were added to a 96-well plate and 10 μL of ciprofloxacin (0, 1, 5, and 10-fold MIC) without or with thymine (10 mM) were added. After incubation at 37°C for 1 h, fluorescence units were immediately measured with the excitation wavelength at 488 nm and emission wavelength at 525 nm using an Infinite M200 Microplate reader (Tecan).
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