Total reactive oxygen species assay kit 520 nm

The reactive oxygen species (ROS) production was measured using CM-H₂DCFDA (C6827, ThermoFisher, Waltham, USA), a cell-permeable probe that is non-fluorescent until oxidation within the cell, and a Total Reactive Oxygen Species (ROS) Assay Kit 520 nm (ThermoFisher). The MCF7 cells were treated with DFP (1 μM to 1000 μM) for 24 to 120 h. The vehicle alone (DMSO) control cells were processed in parallel. After 48 h, the cells were washed with PBS and incubated with CM-H₂DCFDA (diluted in PBS/CM to a final concentration of 1 μM) for 20 min at 37 °C. The same procedure was performed using the Total Reactive Oxygen Species (ROS) Assay Kit 520 nm (ThermoFisher), with an incubation time of 1 h at 37 °C. All subsequent steps were performed in the dark [6 (link)]. The cells were rinsed, harvested, and re-suspended in PBS/CM. The cells were then analyzed by flow cytometry using the SH800 (SONY). The ROS levels were estimated by using the mean fluorescent intensity of the viable cell population. The results were analyzed using the FlowJo software (BD Bioscience, San Jose, CA, USA).

Analysis of the total ROS levels was performed in preparations of the mice red blood cells obtained before the sacrifice. To this purpose a Total Reactive Oxygen Species (ROS) Assay Kit 520 nm (ThermoFisher, Waltham, MA, USA,) was exploited. The cells were resuspended with 100 μL of 1× ROS Assay Stain and incubated for 60 min in a 37°C incubator with 5% CO2. The cells were treated with the desired reagents to induce production of ROS and analyzed on a microplate reader off the 488 nm (blue laser) in the FITC channel.

For quantification of the reactive oxygen species (ROS) levels, the astrocytes (SVGA) and microglia (HMC3) were cultured on 12-well plates (40,000 cells/500 µL medium/well). Before the OGD treatment, the cells were stained using the Total Reactive Oxygen Species (ROS) Assay Kit 520 nm (Cat. 88-5930-74, Thermo Fisher Scientific) according to the manufacturer’s instructions. After 4 h of treatment, the cells were fixed for 20 min in 4% paraformaldehyde (PFA) in PBS at room temperature and washed with PBS, and the dye fluorescence was analyzed using FACSCanto II (Biosciences, Heidelberg, Germany) with 488 nm excitation and detection with a 530/30 band-pass filter. The results were analyzed and plotted using FlowJo™ software v10.7.1 (BD Life Sciences, Heidelberg, Germany).