| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| 25mg |
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| 500mg | |||
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| Targets |
Dihydrorhodamine 123 does not directly target a specific enzyme or receptor. Its oxidation to rhodamine 123 is mediated by H₂O₂ in the presence of peroxidases (e.g. catalase, horseradish peroxidase). [1]
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| ln Vitro |
Increased light production rate is the outcome of stimulating neutrophil NADPH oxidase with 50 nM phorbol 12-myristate 13-acetate (PMA) in the presence of 10 μM dihydrorhodamine 123 (DHR 123). After adding 50 nM PMA, the cells' fluorescence intensity increased over time in the presence of 10 μM dihydrorhodamine 123. When 50 nM PMA was added to induced HL60 cells in the presence of 10 μM dihydrorhodamine 123, the cells' fluorescence increased steadily [1].
In cell‑free systems, Dihydrorhodamine 123 (10 μM) is oxidised slowly by H₂O₂ (147 μM); the rate is greatly enhanced by catalase or horseradish peroxidase, but not by superoxide dismutase. The reaction does not occur without H₂O₂. [1] In phorbol 12‑myristate 13‑acetate (PMA)‑stimulated human neutrophils, Dihydrorhodamine 123 is converted to rhodamine 123. This conversion is insensitive to superoxide dismutase (10 μg/ml) but is inhibited by azide (2 mM) and partially inhibited by diphenylene iodonium (10 μM). External catalase (50 μg/ml) abolishes the fluorescence increase, indicating that H₂O₂ released outside the cell crosses the plasma membrane and reacts with intracellular Dihydrorhodamine 123 in the presence of cellular peroxidases. [1] Uninduced HL60 cells (which lack NADPH oxidase activity) fail to generate rhodamine from Dihydrorhodamine 123 upon PMA stimulation, but do so immediately after addition of H₂O₂. In contrast, induced HL60 cells (expressing NADPH oxidase) show a sustained fluorescence increase after PMA addition. In a 50:50 mixed population of induced and uninduced HL60 cells, both populations become rhodamine‑positive upon PMA stimulation, indicating that H₂O₂ diffuses from producer cells to neighbouring cells. [1] |
| Enzyme Assay |
The oxidation of Dihydrorhodamine 123 to rhodamine 123 was monitored continuously in a fluorimeter at 37 °C in a Na⁺ medium (150 mM NaCl, 1 mM KCl, 5 mM Hepes/Tris, 5.5 mM glucose, pH 7.4). Excitation was at 488 nm and emission at 525 nm. To test cell‑free conversion, 10 μM Dihydrorhodamine 123 was incubated with 147 μM H₂O₂, and the fluorescence increase was recorded. Catalase (15 μg/ml) or horseradish peroxidase (2 μg/ml) was added to assess enhancement. Superoxide dismutase (10 μg/ml) was also tested. No rhodamine generation was observed in the absence of H₂O₂. [1]
Xanthine oxidase was used to generate superoxide; the resulting rhodamine production from Dihydrorhodamine 123 was not inhibited by superoxide dismutase, confirming that H₂O₂ (the dismutation product) is the reactive species. [1] |
| Cell Assay |
Human neutrophils were isolated from buffy coats and resuspended in Na⁺ medium. The conversion of Dihydrorhodamine 123 (10 μM) to rhodamine 123 was monitored in a fluorimeter at 37 °C after stimulation with 50 nM PMA. Inhibitors such as diphenylene iodonium (10 μM or 20 μM), superoxide dismutase (10 μg/ml) and sodium azide (2 mM) were added via an injection port. The rate of rhodamine generation was assessed. [1]
Confocal microscopy was used to image single neutrophils. Cells were settled on glass coverslips in a 750‑μl bath containing 10 μM Dihydrorhodamine 123 at 37 °C. PMA (50 nM) was added and images were collected as Kalman averages of five scans at selected time points. Fluorescence intensity of individual cells increased after PMA addition; this increase was eliminated by 50 μg/ml catalase in the bath. In separate experiments, neutrophils incubated with 1.67 μM rhodamine 123 did not accumulate fluorescence unless 0.2% Triton X‑100 was added, showing that cells are impermeable to rhodamine 123. [1] HL60 cells were cultured in CO₂‑independent medium with 10% foetal calf serum and 2 mM L‑glutamine. Differentiation (NADPH oxidase expression) was induced by 60 mM dimethylformamide for 6 days. Cells (6×10⁶/ml) were incubated with 10 μM Dihydrorhodamine 123 in Krebs‑Ringer buffer at 37 °C, and PMA (50 nM) was added. Rhodamine fluorescence was measured by FACScan (excitation 488 nm, emission 525 nm) using 50‑μl aliquots diluted tenfold. Induced cells showed time‑dependent fluorescence increase, while uninduced cells did not unless H₂O₂ was added. In a 50:50 mixed population (uninduced cells pre‑loaded with 5 μM SNARF‑AM as a marker), both SNARF‑positive and SNARF‑negative cells became rhodamine‑positive after PMA stimulation. [1] |
| References | |
| Additional Infomation |
Dihydrorhodamine 123 is sensitive to light and air; a 2 mM stock solution is prepared in dimethylsulfoxide and stored under nitrogen at −20 °C in the dark. The probe is not suitable for single‑cell or subcellular detection of superoxide because hydrogen peroxide can diffuse from producing cells and oxidise Dihydrorhodamine 123 in any peroxidase‑containing cell, leading to false‑positive identification of non‑producing cells. Only peroxidase‑containing cells fluoresce. The study concludes that Dihydrorhodamine 123 can be used as a probe for superoxide only in bulk cell populations, not at the single‑cell level. [1]
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| Molecular Formula |
C21H18N2O3
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|---|---|
| Molecular Weight |
346.37922
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| Exact Mass |
346.131
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| CAS # |
109244-58-8
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| PubChem CID |
105032
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| Appearance |
White to pink solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
526.9±50.0 °C at 760 mmHg
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| Flash Point |
222.4±26.4 °C
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| Vapour Pressure |
0.0±1.4 mmHg at 25°C
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| Index of Refraction |
1.684
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| LogP |
1.84
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
26
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| Complexity |
488
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
FNEZBBILNYNQGC-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H18N2O3/c1-25-21(24)15-5-3-2-4-14(15)20-16-8-6-12(22)10-18(16)26-19-11-13(23)7-9-17(19)20/h2-11,20H,22-23H2,1H3
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| Chemical Name |
methyl 2-(3,6-diamino-9H-xanthen-9-yl)benzoate
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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: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). This product is not stable in solution, please use freshly prepared working solution for optimal results. |
| 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 : ~100 mg/mL (~288.70 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (7.22 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8870 mL | 14.4350 mL | 28.8700 mL | |
| 5 mM | 0.5774 mL | 2.8870 mL | 5.7740 mL | |
| 10 mM | 0.2887 mL | 1.4435 mL | 2.8870 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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