Quantification of Intracellular ROS Levels

DCF fluorescence helps to measures innumerable ROS such as H₂O₂ and hydroxyl radicals^{49 (link)}. That why DCFDA staining was used to quantify the reactive oxygen species (ROS) level inside the cell as described earlier^{17 (link),41 (link)}. Briefly, the MCF-7 cells (70–80% confluent) were incubated with IC₅₀ concentration of each compound and positive control H₂O_2, respectively in a 24-well culture plates. After 5–6 h incubation of cells with respective compounds, cells were gently washed with 500 μl of prewarmed (at 37 °C) Krebs Ringer buffer (20 mM HEPES, 2 mM MgSO₄, 10 mM dextrose, 127 mM NaCl, 1 mM CaCl₂ and 5.5 mM KCl), subsequently 10 µM DCFDA (Invitrogen Grand Island, NY) has been added to each well and incubated further for 30 min in dark at 37 °C in a humidified CO₂ incubator. After 30 min incubation, the cells were collected by trypsinization and ROS levels were quantified with the help of Flow Cytometry. Nearly 10,000 events for each sample were collected on BD LSR II Flow Cytometry Analyzer, and analyzed with help of FlowJo. For intact cell imaging, cells were washed with PBS, pH 7.4 and imaged for ROS levels estimation using the DCFDA fluorescent dye. Fluorescence images were taken on Nikon-EclipseTS100 microscope.