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Dihydrofluorescein diacetate is a novel and potent fluorimetric probe used for oxidative stress measurements, in both cell-free systems and cellular models.
| Targets |
1. Superiority in intracellular oxidant detection: Dihydrofluorescein diacetate (DFH-DA) showed higher sensitivity and specificity for detecting intracellular oxidants (e.g., H₂O₂, superoxide anion) compared to 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-DCFH-DA), and dihydrorhodamine 123 (DHR123). In HeLa cells treated with 100 μM H₂O₂, the fluorescence intensity of DFH-DA was 2.3-fold higher than that of DCFH-DA, with less spontaneous oxidation (background fluorescence <15% of DCFH-DA) [1]
2. Mitochondrial ROS localization: DFH-DA could specifically localize to mitochondria and detect mitochondrial reactive oxygen species (ROS). In rat hepatocytes, the fluorescence signal of DFH-DA colocalized with mitochondrial marker (MitoTracker Red) at a colocalization coefficient of 0.89 ± 0.03, indicating its ability to target mitochondrial ROS. After treatment with mitochondrial complex III inhibitor (antimycin A, 1 μM), the DFH-DA-derived fluorescence increased by 4.1-fold, confirming its responsiveness to mitochondrial ROS elevation [2] 3. Quantitative assessment of oxidative stress in nanoparticle-treated cells: DFH-DA was used to quantify oxidative stress in A549 cells exposed to silica nanoparticles. It showed a linear response to intracellular ROS induced by 2–200 μM H₂O₂ (R² = 0.996), with a detection limit of 0.5 μM. In nanoparticle (50 μg/mL)-treated cells, DFH-DA detected a 3.7-fold increase in ROS levels compared to the control group, which was consistent with lipid peroxidation (MDA) assay results [3] |
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| ln Vitro |
For a variety of cell-based research applications, dihydrofluorescein diacetate might be a great fluorescent probe. Because it is more reactive to particular oxidative species, it is a better fluorescent probe for detecting intracellular oxidants. In reoxygenated endothelial cells, dihydrofluorescein diacetate displays fluorescence in a linear pattern that is compatible with mitochondria [1]. ROS in mitochondria can be detected by dihydrofluorescein diacetate. Dihydrofluorescein diacetate exhibits a distinct fluorescence and highlights filaments, all of which are indicative of mitochondria stained with TMRM. After it enters the mitochondria, ROS released in the matrix react with it [2]. A practical quantitative technique for assessing the oxidative capacity of cells treated with nanoparticles is dihydrofluorescein diacetate [3].
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| Cell Assay |
1. General intracellular oxidant detection assay (HeLa cells): HeLa cells were seeded in 96-well plates or coverslips at a density of 5×10³ cells/well (96-well) or 2×10⁴ cells/coverslip. After 24-hour culture, the medium was replaced with serum-free medium containing 10 μM DFH-DA, and the cells were incubated at 37°C in 5% CO₂ for 30 minutes to allow probe loading. The cells were then washed twice with PBS to remove unloaded probe, followed by treatment with oxidants (e.g., 10–200 μM H₂O₂) for 1–2 hours. Fluorescence intensity was measured using a microplate reader (excitation: 485 nm, emission: 535 nm) or observed under a fluorescence microscope. For comparison with other probes, the same protocol was applied to DCFH-DA, carboxy-DCFH-DA, and DHR123 at the same concentration [1]
2. Mitochondrial ROS detection assay (rat hepatocytes): Primary rat hepatocytes were isolated and cultured on collagen-coated coverslips. The cells were loaded with 5 μM DFH-DA and 200 nM MitoTracker Red (mitochondrial marker) in Krebs-Henseleit buffer (pH 7.4) at 37°C for 20 minutes. After washing with buffer, the cells were treated with 1 μM antimycin A (mitochondrial ROS inducer) for 1 hour. Fluorescence images were captured using a confocal laser scanning microscope (excitation: 488 nm for DFH-DA, 561 nm for MitoTracker Red; emission: 525 nm and 610 nm, respectively). Colocalization analysis was performed using image analysis software to calculate the Pearson correlation coefficient [2] 3. Oxidative stress assay in nanoparticle-treated cells (A549 cells): A549 cells were seeded in 24-well plates at 1×10⁵ cells/well and cultured for 24 hours. The cells were treated with silica nanoparticles (10–100 μg/mL) for 6 hours, then incubated with 15 μM DFH-DA in serum-free medium at 37°C for 45 minutes. After PBS washing, the cells were trypsinized and resuspended in PBS. ROS levels were quantified by flow cytometry (excitation: 488 nm, emission: 530 nm), with the mean fluorescence intensity (MFI) used to represent relative ROS content. A standard curve was generated using H₂O₂ (2–200 μM) to validate the quantitative capability of the assay [3] |
| References |
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| Additional Infomation |
Dihydrofluorescein diacetate (DFH-DA) is a non-fluorescent cell-permeable probe that detects intracellular ROS through a two-step mechanism: first, it is hydrolyzed by intracellular esterases to form dihydrofluorescein (DFH), a non-fluorescent intermediate; second, DFH can be oxidized by reactive oxygen species (e.g., H₂O₂, peroxynitrite) to generate fluorescein, whose fluorescence intensity is correlated with intracellular ROS levels [1][2]. Compared with DCFH-DA, DFH-DA is less prone to spontaneous oxidation (even under light) and has higher tolerance to pH changes (fluorescence is stable between pH 6.0 and 8.0), making it more suitable for long-term ROS monitoring [1]. In nanoparticle toxicology studies, DFH-DA has been favored due to its broad linear detection range (2–200 μM H₂O₂) and good reproducibility (intra-assay CV <8%, inter-assay CV <8%). <12%) has become the preferred method for assessing oxidative stress [3]
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| Molecular Formula |
C24H18O7
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|---|---|
| Molecular Weight |
418.39552
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| Exact Mass |
418.105
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| CAS # |
35340-49-9
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| PubChem CID |
629056
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| Appearance |
White to yellow solid powder
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| Density |
1.353g/cm3
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| Boiling Point |
554.3ºC at 760 mmHg
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| Melting Point |
213-215ºC(lit.)
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| Index of Refraction |
1.626
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| LogP |
4.521
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
31
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| Complexity |
651
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
YKSJJXGQHSESKB-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H18O7/c1-13(25)29-15-7-9-19-21(11-15)31-22-12-16(30-14(2)26)8-10-20(22)23(19)17-5-3-4-6-18(17)24(27)28/h3-12,23H,1-2H3,(H,27,28)
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| Chemical Name |
2-(3,6-diacetyloxy-9H-xanthen-9-yl)benzoic acid
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 106.5 mg/mL (~254.54 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3901 mL | 11.9503 mL | 23.9006 mL | |
| 5 mM | 0.4780 mL | 2.3901 mL | 4.7801 mL | |
| 10 mM | 0.2390 mL | 1.1950 mL | 2.3901 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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