How to Use Flow Cytometry for Detection of Reactive Oxygen Species (ROS)

Reactive oxygen species (ROS) are key molecules involved in cell signaling, oxidative stress, and apoptosis. Flow cytometry is a powerful tool for measuring intracellular or mitochondrial ROS levels in real time. The following is a step-by-step guide to detecting ROS using flow cytometry:

1. Sample Preparation

Harvest cells from culture or tissue to ensure a single cell suspension. Wash cells with PBS or an appropriate buffer to remove residual culture medium that may interfere with ROS detection.

2. Select a ROS-sensitive probe

Choose a fluorescent probe that reacts with ROS and produces a measurable fluorescent signal. Common probes include: (1) DCF-DA (2',7'-dichlorodihydrofluorescein diacetate): measures general oxidative stress by detecting hydrogen peroxide and hydroxyl radicals. (2) MitoSOX Red: specifically detects mitochondrial superoxide. (3) DHE (dihydroethidium): detects superoxide anion. Follow the manufacturer's instructions to determine probe concentration and incubation time.

3. Staining Procedure

Incubate cells with the ROS probe in the dark at 37°C for 15-30 minutes to allow it to absorb and react with ROS. After incubation, wash cells to remove unbound dye.

4. Flow Cytometry Setup

Configure the flow cytometer with lasers and filters appropriate for the chosen probe (e.g., FITC channel for DCF-DA, PE channel for MitoSOX Red). Use forward scatter (FSC) and side scatter (SSC) to exclude debris and focus on cell populations.

5. Data Acquisition and Analysis

Measure fluorescence intensity to quantify ROS levels. Compare untreated control cells to treated samples to assess oxidative stress levels. Include negative and positive controls (e.g., cells treated with a ROS inducer such as H2O2).

6. Interpret Results

Increased fluorescence intensity indicates elevated ROS levels. Normalize data to untreated controls or baseline fluorescence for comparison.

This method can detect ROS sensitively and specifically, facilitating the study of oxidative stress and related cellular mechanisms.

References

[1] Meghri Katerji et al, Oxid Med Cell Longgev 2019 (doi: 10.1155/2019/1279250)

[2] Shadan Navid et al., Stem Cell Research & Therapy 2017 (DOI:10.1186/s13287-017-0687-y)

[3] Olga Lyublinskaya et al., Redox Biology 2017 (DOI:10.1016/j.redox.2017.04.016)