2. Immunology/Inflammation NF-κB Metabolic Enzyme/Protease
  3. Reactive Oxygen Species
  4. H2DCFDA

H2DCFDA  (Synonyms: DCFH-DA; 2',7'-Dichlorodihydrofluorescein diacetate)

Cat. No.: HY-D0940 Purity: 99.81%
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H2DCFDA (DCFH-DA) is a cell-permeable probe used to detect intracellular reactive oxygen species (ROS) (Ex/Em=488/525 nm).

For research use only. We do not sell to patients.

H2DCFDA Chemical Structure

H2DCFDA Chemical Structure

CAS No. : 4091-99-0

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Customer Review

Based on 457 publication(s) in Google Scholar

H2DCFDA Related Antibodies

Top Publications Citing Use of Products

437 Publications Citing Use of MCE H2DCFDA

IF

    H2DCFDA purchased from MedChemExpress. Usage Cited in: Adv Mater. 2025 Jan 10:e2415030.  [Abstract]

    DCFH-DA (5 μM; 30 min). DCFH‐DA, a specific indicator for ROS detection, is employed to evaluate overall ROS levels in 4T1 cells.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: ACS Nano. 2025 Jan 25.  [Abstract]

    Inside A375 melanoma cells, the ROS level is measured with the help of the DCFH-DA probe under CLSM imaging.
    Representative CLSM images of ROS generation inside A375 cells after different treatments with or without light irradiation (scale bar: 50 μm; blue: Hoechst 33342, green: DCFH-DA probe; Xe lamp, 650 nm, 6.4 mW/cm2, 12 min).

    H2DCFDA purchased from MedChemExpress. Usage Cited in: ACS Nano. 2025 Jan 24.  [Abstract]

    DCFH-DA (10 μM; 30 min). (B) Flow cytometry analysis of ROS levels in HQMitofactories using the DCFH-DA probe. HQ-Mitofactories were exposed to 400 μM H2O2 for 6 h prior to measurement (n = 3; **** is P <0.0001 by one-way ANOVA test). (C) Confocal fluorescent images of ROS levels in HQ-Mitofactories. ROS were labeled with the DCFH-DA probe (green) and nuclei were stained with Hoechst 33342 (blue). Scale bar, 50 μm.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: ACS Nano. 2025 Jan 24.  [Abstract]

    DCFH-DA (10 μM; 30 min). Fluorescent images of ROS levels in PC12 cells. ROS were labeled with the DCFH-DA probe (green) and nuclei were stained with Hoechst 33342 (blue). Scale bar, 40 μm. Data are presented as mean ± SD.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: Adv Mater. 2024 Oct 12:e2411618.  [Abstract]

    DCFH-DA (30 min). The capability of different biocatalysts to eliminate ROS was assessed through c) representative images and d) quantitative analysis (n = 3 independent biological replicates) of DCFH-DA fluorescence across various cell types (MSCs, RSC96, Raw264.7) subjected to distinct treatments.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: Adv Mater. 2024 Nov 26:e2409176.  [Abstract]

    DCFH-DA (10 µm; 20 min). The ROS levels in the oxygen-glucose deprivation/reoxygenation (OGD/R) model of HT22 cells were detected using DCFH-DA. Confocal laser scanning microscope (CLSM) images showed bright green fluorescence in the OGD/R group, indicating a significant increase in ROS.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: Adv Funct Mater. 2024 May 23.

    DCFH-DA (10 µM; 20 min). Fluorescence images of DCFH illustrating intracellular ROS in BMSCs after different treatment (scale bar: 200 µm).

    H2DCFDA purchased from MedChemExpress. Usage Cited in: Bioact Mater. 2024 Jul 9:41:30-45.  [Abstract]

    DCFH-DA (20 min). DCFH-DA assay is further examined to evaluate the ROS scavenging capacity on the cellular level. As ROS oxidizes DCFH into DCF, it emits green fluorescence, which can be used to estimate the remaining ROS.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: J Extracell Vesicles. 2024 Sep;13(9):e12505.  [Abstract]

    H2DCFDA (10 µM; 20 min). Detection of intracellular ROS generation in A549 cells exposed to 20 µM H2O2. Scale bar: 200 µm. Right, quantification of corresponding fluorescence intensity.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: Adv Sci (Weinh). 2022 Mar 10;e2103317.  [Abstract]

    CLSM images of HUVEC cells using DCFH‐DA (0.5 hours) as the fluorescent probe: Control, H2O2, H2O2+10 µg/mL1 PDA NPs, H2O2+40 µg/mL PDA NPs, H2O2+80 µg/mL PDA NPs.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: Acta Pharm Sin B. 16 July 2022.

    Treated HL-1 cells are washed three times and incubated with DMEM medium containing H2DCFDA (10 μM; 30 min) at 37 °C. The cells then are washed three times with warm DMEM medium and intracellular ROS production is detected by flow cytometry.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: Biomaterials. 2022 Jul 21;287:121688.  [Abstract]

    CLSM images of 4T1 tumor cells stained with DCFH-DA (15 min) after different treatments.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: Biomaterials. 2022 May;284:121513.  [Abstract]

    DCFH-DA (10 μM; 30 min). The ROS generation in 4T1 cells and NIH-3T3 cells incubated with various formulations for 4 h observed by CLSM.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: Biomaterials. 2022 Sep 30;290:121842.  [Abstract]

    DCFH-DA (10 μM; 30 min) or C11-BODIPY-labeled cells are substantially increased after treatment with 1 μM FeSO4.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: J Hazard Mater. 2022 Oct 5;439:129502.  [Abstract]

    Representative DCFH-DA (10 μM; 30 min) staining images (Green) in PBNs exposed PSNPs and/or LPS in vivo and in vitro.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: Cell. 2021 Jun 24;184(13):3528-3541.e12.  [Abstract]

    Stained protoplasts with H2DCFDA for the detection of ROS production and PI for PM integrity and tracked the process by live-cell imaging.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: ACS Nano. 2021 Oct 26;15(10):16286-16297.  [Abstract]

    CLSM image of 4T1 cells after being treated with MM, MMP, and MMTP; cells are stained with DCFH-DA (20 μM; for 1 h).

    H2DCFDA purchased from MedChemExpress. Usage Cited in: Biomaterials. 2021 Oct;277:121103.  [Abstract]

    In vitro experiments are conducted to measure the cellular ROS levels in A549 cells from different treatments via flow cytometry using fluorescent probe H2DCFDA (5 μM; 30 min) to better understand the potential molecular mechanism of SOD@ARA290-HBc on radiation-induced lung injury. H2DCFDA fluorescence intensity in A549 cells significantly increased after irradiation.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: Small. 2021 Aug;17(32):e2101368.  [Abstract]

    Laser scanning confocal fluorescence microscopy images of HT-1080 cells treated with different nanodrugs for the detection of intracellular lipid peroxidation and total ROS using C11-BODIPY and DCFH-DA (100 nM; 10 min) as the corresponding fluorescence probes.

    H2DCFDA purchased from MedChemExpress. Usage Cited in: Curr Biol. 2021 May 18;S0960-9822(21)00607-2.  [Abstract]

    Co-staining stigmas with H2DCFDA (50 μM; for 1-2 hours) and the cell wall indicator propidium iodide (PI) shows that ROS is located in the cytoplasm of papilla cells, near the periphery of the plasma membrane.

    • Biological Activity

    • Protocol

    • Purity & Documentation

    • References

    • Customer Review

    Description

    H2DCFDA (DCFH-DA) is a cell-permeable probe used to detect intracellular reactive oxygen species (ROS) (Ex/Em=488/525 nm)[1].

    In Vitro

    Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
    1.1 Preparation of stock solution
    Use anhydrous DMSO to prepare a 10 mM H2DCFDA stock solution.
    Note: It is recommended that the H2DCFDA stock solution be stored at -20 ℃ or -80 ℃ in the dark after aliquoting.
    1.2 Preparation of working solution
    Use preheated serum-free cell culture medium or PBS to dilute the stock solution to prepare a 1-10 μM H2DCFDA working solution.
    Note: Please adjust the concentration of H2DCFDA working solution according to actual conditions and prepare it before use.
    2. Cell staining (suspended cells)
    2.1 Collect cells by centrifugation and wash twice with PBS for 5 minutes each time. The cell density is 1×106/mL.
    2.2 Add 1 mL of dye working solution and incubate at room temperature for 5-30 minutes.
    2.3 Centrifuge at 400 g for 3-4 minutes and discard the supernatant.
    2.4 Add PBS to wash the cells twice, 5 minutes each time.
    2.5 Resuspend the cells in 1 mL of serum-free medium or PBS and observe using a fluorescence microscope or flow cytometer.
    3. Cell staining (adherent cells)
    3.1 Culture the adherent cells on a sterile coverslip.
    3.2 Remove the coverslip from the culture medium and remove the excess culture medium.
    3.3 Add 100 μL of dye working solution, gently shake to completely cover the cells, and incubate for 5-30 minutes.
    3.4 Aspirate the dye working solution, wash 2-3 times with culture medium, 5 minutes each time, and observe using a fluorescence microscope or flow cytometer.
    Note: If flow cytometry is required, the cells need to be digested with trypsin and resuspended before staining.

    MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

    H2DCFDA Related Antibodies
    Molecular Weight

    487.29

    Formula

    C24H16Cl2O7
    CAS No.
    Appearance

    Solid

    Color

    White to off-white

    Emission (Em)

    517
    Excitation (Ex)

    492
    SMILES

    O=C(O)C1=CC=CC=C1C2C3=C(OC4=C2C=C(Cl)C(OC(C)=O)=C4)C=C(OC(C)=O)C(Cl)=C3
    Shipping

    Room temperature in continental US; may vary elsewhere.
    Storage

    -20°C, protect from light

    *In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

    Solvent & Solubility
    In Vitro: 

    DMSO : 125 mg/mL (256.52 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

    Ethanol : 20 mg/mL (41.04 mM; Need ultrasonic)

    Preparing
    Stock Solutions
    Concentration Solvent Mass 1 mg 5 mg 10 mg
    1 mM 2.0522 mL 10.2608 mL 20.5217 mL
    5 mM 0.4104 mL 2.0522 mL 4.1043 mL
    View the Complete Stock Solution Preparation Table

    * Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
    Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

    • Molarity Calculator

    • Dilution Calculator

    Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

    Mass
    =
    Concentration
    ×
    Volume
    ×
    Molecular Weight *

    Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

    This equation is commonly abbreviated as: C1V1 = C2V2

    Concentration (start)

    C1

    ×
    Volume (start)

    V1

    =
    Concentration (final)

    C2

    ×
    Volume (final)

    V2

    In Vivo:

    Select the appropriate dissolution method based on your experimental animal and administration route.

    For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
    To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
    The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

    • Protocol 1

      Add each solvent one by one:  10% DMSO    40% PEG300    5% Tween-80    45% Saline

      Solubility: 2.08 mg/mL (4.27 mM); Suspended solution; Need ultrasonic

      This protocol yields a suspended solution of 2.08 mg/mL. Suspended solution can be used for oral and intraperitoneal injection.

      Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (20.8 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.

      Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
    • Protocol 2

      Add each solvent one by one:  10% DMSO    90% (20% SBE-β-CD in Saline)

      Solubility: ≥ 2.08 mg/mL (4.27 mM); Clear solution

      This protocol yields a clear solution of ≥ 2.08 mg/mL (saturation unknown).

      Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (20.8 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.

      Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.
    In Vivo Dissolution Calculator
    Please enter the basic information of animal experiments:

    Dosage

    mg/kg

    Animal weight
    (per animal)

    g

    Dosing volume
    (per animal)

    μL

    Number of animals

    Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
    Please enter your animal formula composition:
    %
    DMSO +
    % 
    +
    %
    Tween-80 +
    %
    Saline
    Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
    The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).
    Calculation results:
    Working solution concentration: mg/mL
    Method for preparing stock solution: mg drug dissolved in μL  DMSO (Stock solution concentration: mg/mL).

    *In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

    The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).
    Method for preparing in vivo working solution for animal experiments: Take μL DMSO stock solution, add μL . μL , mix evenly, next add μL Tween 80, mix evenly, then add μL Saline.
     If the continuous dosing period exceeds half a month, please choose this protocol carefully.
    Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
    Purity & Documentation

    Purity: 99.82%

    SDS (393 KB)

    COA (252 KB) HNMR (228 KB) LCMS (120 KB)

    Handling Instructions (2659 KB)
    Dyeing Example
    References
    Kinase Assay

    ROS Measurements[1]
    For the detection of intracellular ROS level ,ROS-sensitive probe H2DCFDA is used. Adherent cells (ESCs, difESCs, eMSCs, HeLa, U118) are incubated with 5 µM staining solution in PBS in the dark for 30 min at 37°C, then harvested with 0.05% trypsin-EDTA solution, suspended in a fresh medium, and immediately analyzed with flow cytometer. Lymphocytes, both control and PHA-activated, are resuspended in PBS, incubated with 5 µM of H2DCFDA in the dark for 30 min at 37°C, and immediately analyzed. Along with the H2DCFDA probe, if indicated, ROS-insensitive modification of the fluorescent dye DCFDA is used. The staining procedure is the same as for the H2DCFDA[1].

    MCE has not independently confirmed the accuracy of these methods. They are for reference only.
    References

    Complete Stock Solution Preparation Table

    * Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
    Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

    Optional Solvent Concentration Solvent Mass 1 mg 5 mg 10 mg 25 mg
    Ethanol / DMSO 1 mM 2.0522 mL 10.2608 mL 20.5217 mL 51.3042 mL
    5 mM 0.4104 mL 2.0522 mL 4.1043 mL 10.2608 mL
    10 mM 0.2052 mL 1.0261 mL 2.0522 mL 5.1304 mL
    15 mM 0.1368 mL 0.6841 mL 1.3681 mL 3.4203 mL
    20 mM 0.1026 mL 0.5130 mL 1.0261 mL 2.5652 mL
    25 mM 0.0821 mL 0.4104 mL 0.8209 mL 2.0522 mL
    30 mM 0.0684 mL 0.3420 mL 0.6841 mL 1.7101 mL
    40 mM 0.0513 mL 0.2565 mL 0.5130 mL 1.2826 mL
    DMSO 50 mM 0.0410 mL 0.2052 mL 0.4104 mL 1.0261 mL
    60 mM 0.0342 mL 0.1710 mL 0.3420 mL 0.8551 mL
    80 mM 0.0257 mL 0.1283 mL 0.2565 mL 0.6413 mL
    100 mM 0.0205 mL 0.1026 mL 0.2052 mL 0.5130 mL
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    H2DCFDA Related Classifications

    Help & FAQs
    • Do most proteins show cross-species activity?

      Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

    Keywords:

    H2DCFDA4091-99-0DCFH-DA 2',7'-Dichlorodihydrofluorescein diacetateReactive Oxygen SpeciesInhibitorinhibitorinhibit

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