Measuring ROS Levels in C. jejuni

ROS levels were measured as described previously, with slight modifications (55 (link)). The total ROS accumulation level was measured using the fluorescent dye CM-H₂DCFDA (chloromethyl 2′,7′-dichlorodihydrofluorescein diacetate) (Thermo Fisher Scientific, OH, USA). C. jejuni was prepared as a culture grown overnight on MH agar and resuspended in MH broth to an OD₆₀₀ of 0.1. The bacterial suspension was transferred to a disposable culture tube (Kimble, NJ, USA) and incubated at 4°C. Samples were taken before and after exposure to cold stress for 4 days. After treatment with 10 μM CM-H₂DCFDA dye for 30 min at room temperature, fluorescence was measured using a SpectraMax i3 platform (Molecular Devices, CA, USA) at 495 nm excitation and 527 nm emission wavelengths. The fluorescence levels were normalized to the protein amounts determined using the Bradford assay (Bio-Rad, CA, USA).