Cellular ros assay kit

Effect of aerobic growth of L. reuteri KUB-AC5 was determined. Overnight cultures (0.1% v/v) were transferred into 50 ml MRS broth in 250 ml Erlenmeyer flasks and incubated without aeration (static culture) and with aeration using an orbital shaker at 150 rpm for 24 h. At three-hour intervals, samples were taken for OD₆₀₀ nm and pH measurement, and the growth profile of shaken and non-shaken samples were compared. The samples of cell cultures at early stationary phase (15 h) were collected and analyzed for ROS (Cellular ROS assay kit, ab113851) as well as catalase (Sigma, Catalase assay kit) and SOD activity (Sigma, SOD assay kit). Moreover, the cellular ROS assay was performed by collecting cells in a conical tube with a final OD₆₀₀ of 0.5, then washing with PBS buffer and incubating with 20 μM 2’,7’- dichlorofluorescin diacetate (DCFDA) solution at 37 °C for 30 min in the dark. After that, the cell pellets were washed and were resuspended in PBS buffer by maintaining the same cell concentration. Then, the cell suspension was transferred to a 96 well black/clear bottom microplate and immediately measured for fluorescence intensity using excitation wavelength at 485 nm and emission wavelength at 535 nm. The measured DCF fluorescence intensity was reported in RFU, which represents the ROS generation.