Oct 10, 2024

Public workspaceROS Measurement Using CellROX

DOI
dx.doi.org/10.17504/protocols.io.yxmvme82og3p/v1
  • 1Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA;
  • 2Department of Genetics, Stanford University, Stanford, CA 94305, USA;
  • 3The Institute for Chemistry, Engineering and Medicine for Human Health (Sarafan ChEM-H), Stanford University, Stanford, CA 94305, USA;
  • 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
  • asap
Ali Ghoochani
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DOI: dx.doi.org/10.17504/protocols.io.yxmvme82og3p/v1
External link: https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2Fmp10422.pdf
Protocol Citation: Ali Ghoochani, Monther Abu-Remaileh 2024. ROS Measurement Using CellROX. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvme82og3p/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: October 10, 2024
Last Modified: October 10, 2024
Protocol Integer ID: 109560
Keywords: CellRox, Reactive oxygen species measurement
Abstract
This protocol describes a method to assess oxidative stress levels in cells using the fluorescent probe CellROX using flow cytometry
Materials
  • CellROX-Green reagent (5 µM) (Invitrogen, Cat. No. C10444)
  • PBS
  • Trypsin
  • Flow cytometer
Preparation for Experiment
Preparation for Experiment
Once cells are ready for the experiment, aspirate the media and add fresh media

CellROX Treatment
CellROX Treatment
30m
30m
Treat the cells with 5 µM CellROX-Green (Invitrogen, Cat No. C10444) for Duration00:30:00 at Temperature37 °C

30m
Wash the cells once with PBS.
Cell Detachment
Cell Detachment
3m
3m
Trypsinize the cells to detach them from the culture surface.Duration00:03:00 Temperature37 °C

3m
Centrifuge the cells at Centrifigation1000 rpm Duration00:03:00  and discard the supernatant.

3m
Flow Cytometry Analysis
Flow Cytometry Analysis
Resuspend the cells in PBS.
Measure the intensity of CellROX fluorescence using flow cytometry to assess oxidative stress levels.
Protocol references