Cellular ros assay kit deep red

ROS levels were measured with the Cellular ROS Assay Kit Deep Red (Abcam) following manufacturers’ instructions. Briefly, cells were treated with media containing the indicated concentration of H₂O₂ (Sigma) for 15 min at 37°C in the incubator and stained with1 μl of 1000× ROS Deep Red Stock Solution for additional 30 min at 37°C. Single cells suspension were, analyzed by flow cytometry in PBS with 0.5% BSA and 2 mM EDTA. Data were collected using a BD LSRII flow cytometer. At least 20 000 events were acquired and analyzed using FlowJo software.

ROS levels were assessed by following the instructions of the DCFDA/H2DCFDA‐ Cellular ROS Assay Kit (Abcam, Cambridge, UK) or Cellular ROS assay kit (Deep Red) (Abcam). When applicable, cells were treated with 10 mml‐Glutathione Reduced (Tocris, Bristol, UK) for 18 h before ROS measurement. Ultra pure water was used vehicle control. Fluorescence was measured by the BD Accuri™ C6 Plus flow cytometer (BD Biosciences, San Jose, CA, USA) using the FITC or APC channels.

Cellular ROS Assay Kit (Deep Red) (Abcam, Cambridge, UK) was used for oxidative stress detection. According to the results of the cell viability measurements, 5 and 10 µM of H₂O₂ was used to generate ROS in vitro in the BV-2 cells. A total of 5 × 10³ cells/well were plated for the measurements. The cells were exposed to treatments for 24 h. To check the possible effect of lutein for the ROS generation, freshly added lutein and 24 h of lutein pre-treatment was used for the cells before the H₂O₂ was added. The freshly added lutein was followed by adding H₂O₂ immediately. The assay used an ROS sensor to quantify ROS in live cells. The assay was performed in a 96-well microtiter plate. The experiments were carried out according to the manufacturers’ instructions. Briefly, after 24 h of resting state, the control, the lutein-treated and lutein-pre-treated cells were measured or treated with different concentrations of H₂O₂ to produce ROS and incubated for 10, 20 and 30 min at 37 °C and 5% CO₂. After incubation, the cells were stained and incubated for 30 min at 37 °C and 5% CO₂ with ROS deep red dye solution (100 µL/well). The absorbance was measured using EnSpire Multimode microplate reader (PerkinElmer, Rodgau, Germany) at an excitation wavelength of 650 nm and an emission wavelength of 675 nm with bottom read mode. The ROS changes were determined as a percentage of control.