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| 5mg |
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| Targets |
4,5-Diaminofluorescein (DAF-2) does not have a biological target in the traditional pharmacological sense. Its mechanism of action is chemical rather than biological. DAF-2 reacts with nitric oxide (NO) in the presence of oxygen to form a highly fluorescent triazole derivative. The reaction involves the nitrosation of the diamine groups on the fluorescein ring, followed by cyclization to form the fluorescent product. This reaction is specific for NO and does not occur with other reactive nitrogen species or reactive oxygen species, making DAF-2 a highly selective probe for NO detection.
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| ln Vitro |
4,5-Diaminofluorescein (DAF-2) is essentially non-fluorescent in its native state. Upon reaction with nitric oxide (NO) in the presence of oxygen, it forms a highly fluorescent triazole derivative with excitation and emission maxima at approximately 495 nm and 515 nm, respectively. The fluorescence intensity is proportional to the concentration of NO in the sample. DAF-2 can detect NO concentrations in the nanomolar to micromolar range, making it suitable for measuring physiological NO levels. The compound does not exhibit pharmacological activity; its utility is as a fluorescent indicator for NO.
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| ln Vivo |
4,5-Diaminofluorescein (DAF-2) does not have in vivo pharmacological activity. It is a chemical probe used for research applications, primarily in vitro and ex vivo, to detect and quantify nitric oxide production in biological samples. The compound can be loaded into cells or tissues to measure NO production in real time. However, it is not administered to animals for therapeutic purposes. Its use is limited to research applications for studying NO biology and NOS activity.
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| Enzyme Assay |
Non-cellular assays for 4,5-Diaminofluorescein (DAF-2) involve its use as a fluorescent probe for the detection of NO in cell-free systems. In a typical assay, DAF-2 is dissolved in an appropriate buffer (e.g., PBS, pH 7.4) at a concentration of 5-10 μM. A NO donor such as sodium nitroprusside (SNP), S-nitrosoglutathione (GSNO), or DEA/NONOate is added to generate NO. The fluorescence intensity is measured over time using a spectrofluorometer or a fluorescence microplate reader with excitation at 495 nm and emission at 515 nm. A standard curve is generated using known concentrations of NO donor to quantify NO production.
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| Cell Assay |
In vitro cellular assays for 4,5-Diaminofluorescein (DAF-2) typically involve loading cells with the probe to measure intracellular NO production. Cells are incubated with DAF-2 diacetate (DAF-2 DA), a cell-permeable ester form of the probe, which is hydrolyzed by intracellular esterases to release the active DAF-2. Cells are then stimulated with NO-inducing agents such as lipopolysaccharide (LPS) and cytokines (e.g., IFN-γ) to activate inducible nitric oxide synthase (iNOS), or with calcium ionophores to activate endothelial NOS (eNOS). The increase in fluorescence intensity is measured using a fluorescence microscope, flow cytometry, or a fluorescence microplate reader.
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| Animal Protocol |
4,5-Diaminofluorescein (DAF-2) is not used in in vivo animal studies as a therapeutic or pharmacological agent. Its application is limited to ex vivo analysis of biological samples collected from animals, such as tissue homogenates or plasma, to measure NO production. In some studies, DAF-2 can be administered locally (e.g., by injection into tissues) to measure NO production in vivo, but this is for research purposes only and not for therapeutic evaluation.
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| ADME/Pharmacokinetics |
4,5-Diaminofluorescein (DAF-2) is not a drug and does not have pharmacokinetic properties. Its physical and chemical properties are more relevant: it is a cell-permeable fluorescent probe with a molecular weight of 348.31 and a molecular formula of C₂₀H₁₄N₄O₅. The compound is soluble in DMSO and is typically prepared as a stock solution in DMSO for addition to aqueous buffers or cell culture media. The diacetate form (DAF-2 DA) is used for cell loading due to its increased cell permeability.
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| Toxicity/Toxicokinetics |
Specific toxicological data for 4,5-Diaminofluorescein (DAF-2) are not extensively detailed in standard product information. As a research chemical, it should be handled with standard laboratory precautions, avoiding inhalation, ingestion, and skin contact. The compound is not intended for human use. DAF-2 is generally considered to have low toxicity at the concentrations used for NO detection (typically 5-10 μM). However, higher concentrations may cause cellular toxicity or interfere with cellular functions.
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| References | |
| Additional Infomation |
4,5-Diaminofluorescein is an organic molecular entity whose function is related to dibenzopyran compounds.
4,5-Diaminofluorescein (DAF-2) is a research-grade fluorescent probe intended for laboratory use only and is not approved for clinical use. Its CAS number is 205391-01-1. The primary applications of DAF-2 include the detection and quantification of nitric oxide (NO) in biological systems, measurement of nitric oxide synthase (NOS) activity in cells and tissues, and investigation of NO-mediated signaling pathways. DAF-2 is one of the most widely used fluorescent probes for NO detection due to its high sensitivity, specificity, and cell permeability. The compound is available as the free acid form or as the cell-permeable diacetate ester (DAF-2 DA). |
| Molecular Formula |
C20H14N2O5
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|---|---|
| Molecular Weight |
362.33556
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| Exact Mass |
362.09
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| CAS # |
205391-01-1
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| PubChem CID |
10666340
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| Appearance |
Brown to black solid powder
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| Density |
1.7±0.1 g/cm3
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| Boiling Point |
760.8±60.0 °C at 760 mmHg
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| Flash Point |
413.9±32.9 °C
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| Vapour Pressure |
0.0±2.7 mmHg at 25°C
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| Index of Refraction |
1.848
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| LogP |
1.41
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
27
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| Complexity |
589
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| Defined Atom Stereocenter Count |
0
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| SMILES |
0
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 2.7598 mL | 13.7992 mL | 27.5984 mL | |
| 5 mM | 0.5520 mL | 2.7598 mL | 5.5197 mL | |
| 10 mM | 0.2760 mL | 1.3799 mL | 2.7598 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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