Apr 20, 2026
CellROX Green Live Imaging Protocol
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Elena Coccia 2026. CellROX Green Live Imaging Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9bo64l3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 28, 2026
Last Modified: April 20, 2026
Protocol Integer ID: 244170
Keywords: CellROX Green, reactive oxygen species, live cell imaging, oxidative stress, time imaging of oxidative stress, green live imaging protocol, green live imaging protocol this protocol, cellrox green reagent, levels in live cell, live cell, cellular environment
Abstract
This protocol is designed to assess reactive oxygen species (ROS) levels in live cells using the CellROX Green reagent, enabling real-time imaging of oxidative stress in cellular environments.
Materials
Complete Culture Medium (DMEM without phenol red and riboflavin, supplemented with 10% FBS and 1% pen/strep) - 10 mL per well
CellROX Green Reagent (2.5 mM in DMSO) - 10 µL (for 1:1000 dilution in 10 mL medium)
Phosphate Buffered Saline (PBS) (pH 7.4) - 10 mL (for washing)
96-well plastic-bottom plates (µ clear; Greiner Bio-One) - 1 plate
Reagents:
Fluorescent Dye CellROX Green - A cell-permeable probe that fluoresces upon oxidation by ROS
Troubleshooting
Problem
Low fluorescence signal
Solution
Ensure proper dilution of CellROX Green; check microscope settings for optimal exposure.
Problem
High background fluorescence
Solution
Ensure cells are washed thoroughly with PBS to remove excess dye.
Safety warnings
Handle all reagents with care, using appropriate personal protective equipment (PPE). Dispose of all waste according to institutional safety guidelines.
Preparation of Working Stain
Warm complete culture medium to 37 °C. Dilute CellROX Green reagent directly into the culture medium , resulting in a final concentration of 1µM.
Note: for microglia we change half medium only, so the conentration of the solution will be 2µM, to have 1µM as final concentration on cells
Staining Live Cells
Add media to cells.
Incubation
Incubate the cells at 37 °C for 30 minutes, protected from light.
Washing / Medium Change
Remove the staining medium. Replace with fresh, pre-warmed medium.
Live Imaging (ROS Readout)
Image the cells immediately using an epifluorescence or confocal microscope with a FITC/GFP-like channel (excitation ~485 nm, emission ~520 nm).
Imaging
Capture images of the cells to assess ROS levels.
Protocol references