Jan 21, 2025
Measuring ROS production by neutrophils using DCFH-DA probe and flow Cytometry
- 1Faculty of Health Sciences
Protocol Citation: Valeria Duarte de Almeida 2025. Measuring ROS production by neutrophils using DCFH-DA probe and flow Cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj9j3blk5/v1
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Protocol status: Working
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Created: January 21, 2025
Last Modified: January 21, 2025
Protocol Integer ID: 118815
Keywords: ROS, Neutrophils
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Abstract
Neutrophils are isolated, stained with the DCFH-DA probe, and incubated with or without stimuli (e.g., PMA or fMLP) to induce ROS production. After incubation, fluorescence is measured by flow cytometry to assess intracellular ROS levels. Stimulated neutrophils should exhibit increased fluorescence intensity compared to unstimulated controls, indicating higher ROS production. The presence of inhibitors like DPI should result in a significant reduction in fluorescence, confirming the specificity of ROS detection.
Guidelines
To ensure the accuracy, reproducibility, and safety of the experiment, the following guidelines should be followed:
- Use anticoagulants like EDTA or heparin to prevent clotting.
- Process blood samples promptly (within 2–4 hours) to ensure neutrophil viability.
- Ensure >95% cell viability using trypan blue or propidium iodide staining before proceeding with the assay.
- Handle cells gently to avoid activation or damage.
- Always include appropriate controls (negative control, positive control with PMA, and inhibitor control with DPI) to validate the experiment.
- Use biological replicates (at least 3) to ensure statistical significance.
- Maintain consistent cell concentrations across samples (e.g., 1 × 10⁶ cells/mL).
- Use freshly prepared reagents to prevent degradation and variability.
- Prepare DCFH-DA solutions under low light conditions to prevent premature oxidation.
- Aliquot reagents to avoid repeated freeze-thaw cycles.
- Set appropriate forward and side scatter parameters to properly gate neutrophil populations.
- Conduct experiments under identical conditions, such as temperature, incubation times, and reagent batches.
- Use standardized protocols for sample preparation and data analysis.
- Work in a sterile environment to prevent bacterial contamination, which may alter results.
- Regularly clean flow cytometry fluidics to avoid cross-contamination.
Materials
- Neutrophils suspension (isolated from human peripheral blood or animal models)
- DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) probe
- Phosphate-buffered saline (PBS), pH 7.4
- RPMI 1640 medium (with 10% fetal bovine serum, optional)
- Stimuli for positive control: Phorbol 12-myristate 13-acetate (PMA)
- Inhibitors (if needed): Diphenyleneiodonium (DPI, NADPH oxidase inhibitor)
- Flow cytometer with excitation/emission filters for FITC (488/530 nm)
- FACS tubes or 96-well plates
- Refrigerated centrifuge and 37°C water bath
Safety warnings
- Biological Hazards: Handle blood-derived neutrophils with care to avoid potential infections. Disinfect work surfaces before and after the experiment.
- Chemical Hazards: PMA and DPI are potentially hazardous; handle them inside a fume hood and dispose of them properly.
- Laser Safety: Avoid direct exposure to the laser beams of the flow cytometer to prevent eye damage.
Ethics statement
Human Samples:
- Obtain informed consent from donors in compliance with institutional and national ethical regulations.
- Ensure anonymity and confidentiality of donor data.
- Dispose of biohazardous waste following institutional protocols.
Animal Studies:
- Conduct experiments according to ethical guidelines outlined by relevant regulatory bodies (e.g., ARRIVE, IACUC).
- Use the minimum number of animals required to achieve statistical power.
If approval was obtained, please include the name of the IACUC or equivalent ethics committee and any relevant permit numbers.
Before start
Before starting the experiment, ensure the following conditions are met:
- Personal Protective Equipment (PPE): Wear gloves, lab coat, and safety goggles when handling blood samples and reagents.
- Biosafety: Follow biosafety level 2 (BSL-2) guidelines if working with human blood or primary cells.
- Instrument Calibration: Verify that the flow cytometer is properly calibrated and compensated to avoid false-positive signals.
- Reagent Preparation: Prepare fresh DCFH-DA working solutions to prevent auto-oxidation and ensure accurate results.
- Sample Collection Ethics: Ensure all human samples are collected with informed consent and under the approval of an Institutional Ethics Committee.
Neutrophil Isolation and Preparation
Neutrophil Isolation and Preparation
3h 30m
3h 30m
Isolate neutrophils from whole blood using density gradient centrifugation (e.g., Ficoll-Paque).
3h
Wash cells twice with PBS to remove any residual isolation medium.
Count the cells and adjust the concentration to approximately 1 × 10⁶ cells/mL in RPMI 1640 or PBS.
30m
Staining with DCFH-DA
Staining with DCFH-DA
45m
45m
Prepare the DCFH-DA working solution at a final concentration of 5–10 µM in PBS.
5m
Add the DCFH-DA to the neutrophil suspension and incubate for 20–30 minutes at 37°C in the dark, allowing the probe to penetrate the cells and be hydrolyzed to non-fluorescent DCFH.
30m
After incubation, wash cells with PBS to remove excess probe.
10m
ROS Stimulation and Measurement
ROS Stimulation and Measurement
1h 20m
1h 20m
Divide cells into different experimental groups, such as:
- Unstimulated control (no treatment)
- Positive control (treated with 100 nM PMA)
- Experimental groups (treated with stimuli of interest, e.g., fMLP)
- Inhibitor groups (pretreated with DPI before stimulation)
Incubate cells with the desired stimulus for 30–60 minutes at 37°C.
1h
After incubation, immediately place cells on ice to stop the reaction
Analyze fluorescence using a flow cytometer, with excitation at 488 nm and emission at 530 nm (FITC channel).
20m
Flow Cytometry Analysis
Flow Cytometry Analysis
20m
20m
Set up the flow cytometer using unstained cells (gate) to establish background fluorescence.
2m
Acquire at least 10,000 events per sample.
Analyze data in terms of:
- Mean fluorescence intensity (MFI), which reflects the level of ROS production.
- Percentage of positive cells producing ROS.
18m
Compare results across conditions to determine changes in ROS levels.
Data Analysis and Interpretation
Data Analysis and Interpretation
- Increased fluorescence indicates higher ROS production.
- Compare MFI values between treated and control samples.
- Use statistical tests (e.g., ANOVA or t-test) to assess significance.
Protocol references
Ito, Y., Lipschitz, D.A. (2002). Assay of Intracellular Hydrogen Peroxide Generation in Activated Cytometry Individual Neutrophils by Flow. In: Armstrong, D. (eds) Oxidants and Antioxidants. Methods in Molecular Biology‱, vol 196. Humana Press. https://doi.org/10.1385/1-59259-274-0:111
Eruslanov, E., Kusmartsev, S. (2010). Identification of ROS Using Oxidized DCFDA and Flow-Cytometry. In: Armstrong, D. (eds) Advanced Protocols in Oxidative Stress II. Methods in Molecular Biology, vol 594. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-411-1_4
Muñoz-Sánchez G, Godínez-Méndez LA, Fafutis-Morris M, Delgado-Rizo V. Effect of Antioxidant Supplementation on NET Formation Induced by LPS In Vitro; the Roles of Vitamins E and C, Glutathione, and N-acetyl Cysteine. Int J Mol Sci. 2023 Aug 24;24(17):13162. doi: 10.3390/ijms241713162. PMID: 37685966; PMCID: PMC10487622.