Optimized, automated assays for HCS

Generation of reactive oxygen species (ROS) is inevitable for aerobic organisms and, in healthy cells, occurs at a controlled rate. Increased production of ROS by a cell is an early hallmark of cellular stress. Intracellular ROS cause damage to proteins, nucleic acids, and membrane lipids. Oxidative damage of these biomolecules is associated with aging as well as a variety of pathological events, including atherosclerosis, carcinogenesis, ischemia reperfusion injury, and neurodegenerative disorders. Using fluorogenic reporters such as CellROX dyes allows for the real-time analysis of ROS generation in living cells and in fixed-cell preparations.

Typical oxidative stress assay

Live or fixed-cell assay protocol.

Imaging mode

  • Widefield or confocal
  • 10x or 20x magnification

Automatically measured properties

  • Fluorescence intensity, morphology, and count values for each object
  • Fluorescence intensity, morphology, and count values of spots in different cell regions
  • Average fluorescence intensity difference and ratios between different regions for each cell
  • Intensity and count ratio between channels within different regions for each cell

Oxidative stress assay images

Hap1 cells were treated with menadione and labeled with CellROX Green and Hoechst 33342. The cells were imaged using a Thermo Scientific CellInsight CX5 high-content platform (row A), and Thermo Scientific HCS Studio Software, which were used to automatically identify (row B) and quantify the intensity of CellROX green fluorescence from each cell.


Sample experimental data

Dose-dependent increase in ROS production in Hap 1 cells loaded with CellROX Green and treated with menadione for one hour.