Reactive oxygen species (ROS) are by-products of cellular metabolism, including superoxide anion radicals, hydrogen peroxide, hydroxyl radicals, etc. They play roles in cellular signaling, but excessive production can lead to cellular damage and is associated with various diseases. Current methods for detecting ROS include fluorescent staining, chemiluminescence, electron paramagnetic resonance (EPR/ESR), electrochemical biosensors, chromatography, spectrophotometry, and methods based on fluorescent proteins (Table 1).

 

Table 1 Common Methods for ROS Detection

ROS Detection Method	Method Description
Fluorescent Staining	Using specific fluorescent probes such as DCFH-DA, which can detect various ROS. The probe itself is non-fluorescent, enters cells and is hydrolyzed by esterases to generate DCFH, which is then oxidized by ROS to produce the fluorescent DCF.
Chemiluminescence	Utilizing the luminescent signals generated from the reaction of certain chemicals with ROS for quantitative detection of ROS.
Electron Paramagnetic Resonance (EPR/ESR)	A direct method for detecting free radicals, suitable for detecting various ROS including hydroxyl radicals.
Electrochemical Biosensors	Detection of ROS using electrochemical methods, characterized by high sensitivity and selectivity.
Chromatography	Detection of specific ROS or their metabolites through chromatographic separation techniques.
Spectrophotometry	Using specific spectrophotometers to detect ROS, such as detecting superoxide anion by monitoring the reduction of nitroblue tetrazolium (NBT).
Fluorescent Protein-based Methods	Genetic engineering techniques are used to fuse fluorescent proteins with ROS-sensitive peptides, and ROS are detected through changes in fluorescence.

 

Detection of ROS levels is crucial for understanding cellular physiological functions and cell damage caused by environmental factors. Today, we will focus on the principles and specific operational steps of detecting intracellular ROS using fluorescent staining.

 

ROS Detection Guide

 

I. Selection of Appropriate Probes

 

DCFH-DA: This is a commonly used fluorescent probe that quantifies intracellular ROS levels by detecting the fluorescence intensity of its oxidation product DCF. DCFH-DA itself is non-fluorescent, can freely cross cell membranes, is hydrolyzed by esterases within cells to generate DCFH, and is then oxidized to produce DCF, with fluorescence intensity being proportional to ROS levels.

 

Figure 1 Mechanism of action of DCFH-DA probe in cells^[1]

 

II. Experimental Steps (Taking abs580232, ROS Detection Kit as an example)

 

1. Loading ROS Probes

 

1) In situ Probe Loading (Only applicable to adherent cells)

① Cell Preparation: Plate cells the day before detection to ensure that the cell density is less than 5×10⁵/mL at the time of detection.

② Drug Induction: Remove the cell culture medium, add drug treatment diluted in serum-free medium, and incubate in the cell culture incubator at 37℃ in the dark. The actual induction time is determined by the drug characteristics and cell type.

③ (Optional) Positive Control: Dilute the positive control (abs580232, Rosup, 100mM) with serum-free medium to a commonly used working concentration of 100μM, add to cells, and incubate at 37℃ in the dark for 30min-4h to increase ROS levels. Different cell types may vary. For example: HeLa cells require 30-60min of incubation, while MRC5 human embryonic fibroblasts require 90min.

④ ROS Probe Preparation: Dilute DCFH-DA 1:1000 with serum-free culture medium before probe loading to achieve a final concentration of 10μM.

⑤ ROS Probe Loading: Remove the drug treatment, add the appropriate volume of the diluted DCFH-DA working solution. The volume added should fully cover the cells. For example: for a 6-well plate, at least 1mL is usually required, and for a 96-well plate, at least 100μL is usually required. Incubate at 37℃ in the cell culture incubator in the dark for 30min.

⑥ Cell Washing: Wash the cells 1-2 times with serum-free culture medium to thoroughly remove DCFH-DA that has not entered the cells.

 

2) Probe Loading After Cell Collection (Applicable to both adherent and suspension cells)

① Cell Preparation: Culture cells according to standard methods, ensuring the cell condition for detection. Collect a sufficient amount of cells using appropriate methods.

② Drug Induction: Suspend the collected cells in an appropriate volume of diluted drug and incubate in the cell culture incubator at 37℃ in the dark. The actual induction time is determined by the drug characteristics and cell type.

③ (Optional) Positive Control: Dilute the positive control (abs580232, Rosup, 100mM) with serum-free medium to a commonly used working concentration of 100μM, add to cells, and incubate at 37℃ in the dark for 30min-4h to increase ROS levels. Different cell types may vary. For example: HeLa cells require 30-60min of incubation, while MRC5 human embryonic fibroblasts require 90min.

④ ROS Probe Preparation: Dilute DCFH-DA 1:1000 with serum-free culture medium before probe loading to achieve a final concentration of 10μM.

⑤ Probe Loading: Remove the drug from the cells, collect the cells by centrifugation, and add the diluted probe to achieve a cell density of 1.0×10⁶-2.0×10⁷. Note: The cell density should be adjusted according to the subsequent detection system, detection method, and total detection volume. For example: for flow cytometry, the number of cells per tube should be no less than 10⁴ and not more than 10⁶.

⑥ Cell Washing: Wash the cells 1-2 times with serum-free cell culture medium to thoroughly remove DCFH-DA that has not entered the cells.

 

2. Fluorescence Microscopy Detection

 

1) For adherent cells or live tissues, direct observation under the fluorescence microscope is possible; for suspension cells, take 25-50μL of cell suspension and drop it onto a microscope slide, then cover with a cover slip.

2) Under the fluorescence microscope, use the FITC filter to observe fluorescence and remove background to observe changes in fluorescence.

 

3. Flow Cytometry Analysis

 

1) For adherent cells, digest with trypsin to prepare a single-cell suspension; for suspension cells, collect the cells directly. Resuspend the cells with 0.5-1mL of PBS (0.5-1×10⁵/mL).

2) Select the FL1 or BL1 channel of the flow cytometer, excite at 488nm, and measure the emission at 530nm. Cells should be divided into two subpopulations: ROS-negative cells with very low fluorescence intensity and ROS-positive cells with strong green fluorescence.

 

Application of DCFH-DA in ROS Detection

 

Gao^[2] et al. used the standard fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate to measure ROS in 4T1 cells after liposome treatment. The specific procedure is as follows:

1) 4T1 cells were seeded in 35mm dishes at a density of 8×10⁴ cells/well.

2) After 24h of cell culture with a confluence of 70–80%, liposomes (1mL, 1mg/mL) were added to the culture medium. After 6h of treatment, cells were washed three times with PBS.

3) Cells were stained with DCFH-DA (10μM), incubated for 30min, washed three times with PBS, and then imaged using confocal laser scanning microscopy (CLSM).

Figure 2 ROS Imaging in 4T1 Cells^[2]

 

Regardless of the liposome type, fucosylated liposomes generated more ROS in 4T1 cells than non-targeted liposomes (Figure 2), mainly due to enhanced cell uptake. Due to DAPC peroxidation, DAPC-Fuc was more effective in ROS generation than DOPC-Fuc.

 

References:

[1] Rajneesh, Pathak J, Chatterjee A, et al. Detection of Reactive Oxygen Species (ROS) in Cyanobacteria Using the Oxidant-sensing Probe 2',7'-Dichlorodihydrofluorescein Diacetate (DCFH-DA)[J]. Bio Protoc. 2017;7(17):e2545.

[2] Gao Z, Zhang J, Hou Y, et al. Boosting the synergism between cancer ferroptosis and immunotherapy via targeted stimuli-responsive liposomes[J]. Biomaterials.2024;305:122442.

 

Recommended ROS Detection Products:

Catalog Number	Product Name	Specifications
abs580232	Reactive Oxygen Species (ROS) Detection Kit (Green Fluorescence)	1000T
abs580233	Reactive Oxygen Species (ROS) Detection Kit (Red Fluorescence)	100-500T

 

Recommended Oxidative Stress Related Products:

Detection Item	Catalog Number-Specifications	Product Name	Detection Method/Range
Lipid Peroxidation Products	abs580011-96T	Malondialdehyde (MDA) Detection Kit	Colorimetric; 0.01mmol/L-1mmol/L
Antioxidant Enzymes	abs580010-96T	Superoxide Dismutase (SOD) Detection Kit	Colorimetric; 1U/mL-30U/mL
Glutathione Peroxidase Detection Kit	abs580136-96T	Glutathione Peroxidase Detection Kit	Colorimetric; 0.01mmol/L-5mmol/L
Glutathione Reductase Detection Kit	abs580042-96T	Glutathione Reductase Detection Kit	Colorimetric; 4umol/L-400umol/L
Glutathione S-Transferase (GST) Detection Kit	abs580046-96T	Glutathione S-Transferase (GST) Detection Kit	Colorimetric
Non-enzymatic Antioxidants	abs580006-96T	Glutathione Detection Kit	Colorimetric; 0.01mmol/L-0.5mmol/L
Vitamin E Detection Kit	abs580153-96T	Vitamin E Detection Kit	Colorimetric; 50umol/L-2000umol/L
Vitamin C Detection Kit	abs580047-96T	Vitamin C Detection Kit	Colorimetric; 0.02mmol/L-2mmol/L
Coenzyme Q10 Detection Kit	abs580226-96T	Coenzyme Q10 Detection Kit	Colorimetric; 0.1mmol/L-10mmol/L
Total Antioxidant Capacity Detection Kit	abs580109-96T	Total Antioxidant Capacity Detection Kit	Colorimetric; 0.04mmol/L-4mmol/L

 

Recommended Mitochondrial Oxidative Damage Detection Products:

Catalog Number	Product Name	Specifications
abs580238	Mitochondrial Respiratory Chain Complex I Activity Detection Kit (Micro Method)	96T
abs580239	Mitochondrial Respiratory Chain Complex II Activity Detection Kit (Micro Method)	96T
abs580240	Mitochondrial Respiratory Chain Complex III Activity Detection Kit (Micro Method)	96T
abs580241	Mitochondrial Respiratory Chain Complex IV Activity Detection Kit (Micro Method)	96T
abs580242	Mitochondrial Respiratory Chain Complex V Activity Detection Kit (Micro Method)	96T
abs50064	Mitochondrial Permeability Transition Pore (MPTP) Detection Kit	50T

 

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