Quantifying Cellular ROS in L. plantarum

L. plantarum ST-III was cultivated in TYC medium under AE or AN conditions, as described above. After 12 h cultivation, bacteria cells were harvested by centrifugation at 5,000 rpm for 2 min and the level of reactive oxygen species (ROS) was determined using 2,7-dichlorodihydrofluorescein diacetate (H₂DCF-DA; Beyotime Institute of Biotechnology, Haimen, China) according to the instructions provided by the manufacturer. In brief, around 1 × 10⁶ cells were collected and washed with phosphate-buffered solution (0.01 M phosphate, pH 7.4, PBS) and then treated with 10 mM H₂DCF-DA dissolved in PBS at 37°C anaerobically for 20 min. After removal of H₂DCF-DA and three times wash with PBS, the fluorescence intensity was monitored with excitation wavelength at 488 nm and emission wavelength at 525 nm on SpectraMax M5, Molecular Devices (San Jose, CA, United States).
For each sample, an equal amount of cells were sonicated and subjected to quantification of the total protein using the Bradford method. The fluorescence intensity was normalized with the total protein content, and the relative amount of ROS is expressed as DCF fluorescence intensity per milligram total protein, as described previously (Yan et al., 2018b (link)). The experiments were performed in triplicate and the average values, as well as the standard deviations, are shown.