Flow Cytometry Assay to Quantify Oxidative Stress: An Assay to Quantify Reactive Oxygen Species in Intestinal Organoids Using Fluorogenic Probes
JoVE Encyclopedia of Experiments
Biological Techniques
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Intestinal cells produce reactive oxygen species - ROS. Moderate ROS levels maintain cellular homeostasis, while high ROS levels can cause cell death.
To quantify ROS levels in vitro, begin with a culture of mouse intestinal 3D organoids. These organoids exhibit gut-epithelium architecture having crypt-like protrusions harboring green fluorescence-expressing stem cells. They also contain villus-like differentiated cells at the periphery, surrounding the central lumen comprising dead cells and debris. All these cells produce different levels of ROS.
Now, treat the organoids in the designated wells with ROS modulators. Based on their nature, modulators can act as ROS inducers or inhibitors. After incubating for the required duration, remove the spent medium.
Add a proteolytic enzyme to the culture, disrupting the organoids' 3D architecture and releasing different cells in suspension. Next, add a ROS-sensitive fluorogenic probe and incubate.
Upon entering the cells, the probes react with ROS and get oxidized to produce red fluorescence. Treat the cells with DAPI - a DNA-binding fluorescent dye - which selectively stains the nuclei of dead cells - creating a contrast with the live cells.
Using a flow cytometer, analyze the DAPI-negative live cells to obtain a red fluorescence signal corresponding to their ROS level. Depending on the nature of modulators, inducers increase ROS levels while the inhibitors decrease them.
Add 1 microliter of the N-acetyl cysteine stock solution in the negative control wells of the 96-well round-bottom plate containing the organoids.
After a 1-hour incubation, add 1 microliter Tert-butyl hydroperoxide stock solution in the corresponding wells, and incubate for 30 minutes. Then, using a multi-channel pipette, remove the medium without disturbing the attached BMM, and transfer it to another 96-well round-bottom plate. Keep this plate aside.
Next, add 100 microliters of trypsin, and using a multi-channel pipette, pipette up and down five times to destroy the BMM. After a short 5-minute incubation, dissociate the organoids by pipetting up and down a second time.
Spin the plate, and discard the supernatant by inverting the plate. Add the medium previously collected in another 96-well plate, back into the corresponding wells, and re-suspend the cells by pipetting up and down five times.
Next, add 1 microliter of the fluorogenic probe, and incubate for 30 minutes. Then, spin the plate again, and re-suspend the cells with 250 microliters of DAPI solution. Transfer the samples in Flow cytometry tubes, and keep the tubes on ice.
Optimize the forward and side scatter voltage settings on unstained control and laser voltages, for each fluorophore using mono-stained samples. Then, using an appropriate gating strategy, collect a minimum of 20,000 events.
Overview
This study investigates the production of reactive oxygen species (ROS) in mouse intestinal 3D organoids. It explores the effects of ROS modulators on cellular homeostasis and cell viability.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Oxidative Stress
Background
- Intestinal cells produce varying levels of ROS.
- Moderate ROS levels are essential for cellular homeostasis.
- Excessive ROS can lead to cell death.
- Understanding ROS dynamics is crucial for gut health.
Purpose of Study
- To quantify ROS levels in intestinal organoids.
- To assess the impact of ROS inducers and inhibitors.
- To analyze cell viability in relation to ROS levels.
Methods Used
- Culture of mouse intestinal 3D organoids.
- Treatment with ROS modulators.
- Flow cytometry analysis of ROS levels using fluorogenic probes.
- Use of DAPI for distinguishing live and dead cells.
Main Results
- ROS levels varied among different cell types in organoids.
- Inducers increased ROS levels, while inhibitors decreased them.
- Flow cytometry effectively quantified ROS in live cells.
- Cell viability was assessed through DAPI staining.
Conclusions
- ROS play a critical role in intestinal cell function.
- Modulators can effectively alter ROS levels in organoids.
- This method provides insights into oxidative stress in gut cells.
Frequently Asked Questions
Executive Industry Relevance
This flow cytometry assay enables quantitative measurement of reactive oxygen species in 3D intestinal organoids, providing a physiologically relevant model for evaluating compound effects on oxidative stress pathways. It supports target validation and mechanistic de-risking in early discovery by linking ROS modulation to cellular outcomes in a disease-relevant system. The assay delivers quantitative, reproducible data that can inform go/no-go decisions in lead identification and preclinical prioritization.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses by quantifying ROS changes in response to modulators, clarifying pathway involvement in oxidative stress.
- Operational Value: Enables functional validation of targets linked to ROS regulation using a disease-relevant intestinal organoid model.
- Predictive Value: Supports portfolio triage by providing dose-responsive, quantitative readouts that correlate compound activity with biological effect.
Screening & Assay Development
- Assay Readiness: Generates standardized, quantitative fluorescence outputs from dissociated organoids suitable for high-throughput flow cytometry screening.
- Reproducibility: Uses gated analysis of DAPI-negative live cells to ensure consistent ROS signal measurement across experiments.
- Scalability: Compatible with 96-well plate formats and multi-channel pipetting, enabling parallel compound testing.
Translational & Preclinical Research
- Disease Relevance: Models human intestinal oxidative stress using murine organoids with gut-epithelium architecture and differentiated/villus-like structures.
- Translational Continuity: Bridges discovery and preclinical work by providing a quantifiable biomarker (ROS flux) for mechanism-of-action confirmation.
- Risk-Adjusted Decisions: Enables evaluation of compound-induced oxidative stress as a safety or efficacy signal prior to in vivo studies.
Pipeline & Workflow Integration
The assay fits within the discovery continuum from target validation through lead identification to preclinical assessment, offering a quantitative oxidative stress readout that informs compound progression.
- Discovery Biology: Supports hypothesis testing by measuring ROS as a functional output of pathway modulation in intestinal epithelial cells.
- Screening: Delivers assay-ready, quantitative fluorescence data enabling reliable comparison of compound effects on ROS levels.
- Analytics: Provides flow cytometry-based quantification of red fluorescence in live cells, allowing statistical comparison of ROS induction or inhibition.
- Translational Research: Connects to preclinical work through a measurable, disease-relevant oxidative stress phenotype that can be monitored across models.
- Enterprise Reuse: Establishes a standardized, transferable method for oxidative stress assessment applicable across multiple GI-targeted programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by linking compound treatment to quantifiable ROS changes in a physiologically relevant model.
- Operational Value: Delivers standardized, reproducible workflow from organoid culture to flow cytometry readout, minimizing variability.
- Strategic Value: Improves go/no-go decisions by providing early mechanistic insight into oxidative stress liability or efficacy.
- Portfolio Impact: Enables risk-adjusted prioritization of compounds based on their effect on ROS homeostasis in disease-relevant tissue.
Implementation Considerations
- Requires expertise in 3D organoid culture, flow cytometry, and fluorescent probe handling.
- Dependent on access to flow cytometer with appropriate fluorescence channels and compensation controls.
- Necessitates standardization of organoid dissociation and staining protocols across users and sites.
- Must account for variability in organoid size and differentiation state when interpreting ROS signals.
- Limited to ex vivo models; does not capture systemic metabolism or microbiome influences on ROS.
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Last updated: 29 August 2026