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| Targets |
TRFS-red selectively targets thioredoxin reductase (TrxR), a crucial selenoprotein in the cellular antioxidant defense system. The probe's structure includes a disulfide bond that is specifically reduced by the selenocysteine residue in the active site of TrxR. This reduction triggers an intramolecular cyclization reaction, which releases a highly fluorescent naphthalimide molecule. The spectral properties of TRFS-red are tuned for in vivo and in vitro imaging, allowing for the real-time monitoring of TrxR activity in living systems.
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| ln Vitro |
In living HeLa cells, TRFS-red (1 μM; 30-120 min) produces a red fluorescence signal, with the majority of the red fluorescence occurring in the cytosol[1]. In TEBU, TRFS-red (10 μM) has a maximum absorption at about 530 nm.(pH 7.4) with 50 mM Tris-HCl and 1 mM EDTA[1]. A dependable fluorescence signal is displayed by TRFS-red in the pH range of 5.5–8.5[1].
In vitro activity is demonstrated as a sensitive and selective sensor for TrxR activity. TRFS-red exhibits a high response rate and sensitivity to TrxR when tested in cell-free systems containing the purified enzyme. When the probe is incubated with recombinant TrxR in the presence of its cofactor NADPH, a strong, concentration-dependent increase in red fluorescence is observed. The probe shows minimal response to other common biological reductants, such as glutathione (GSH) and cysteine, confirming its high selectivity for TrxR (Sec dependency) [25L22-L24] [3L36-L37]. |
| ln Vivo |
TRFS-red is specifically designed for live-cell imaging to visualize TrxR activity in its native environment. When applied to living cells, TRFS-red has been shown to be cell-permeable and produce a red fluorescence signal that is predominantly distributed in the cytosol, where the main isoform of TrxR is located. HeLa cells treated with 1 microM TRFS-red for 30 to 120 minutes exhibit a strong red fluorescence signal, indicating active reduction of the probe by the cellular TrxR enzyme. This signal is significantly reduced when the cells are pretreated with known TrxR inhibitors like auranofin or DNCB, confirming its specificity [25L26-L27].
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| Enzyme Assay |
The enzymatic activity assay for TrxR using TRFS-red can be performed in 96-well plate format. To a reaction mixture containing 100 uL of 50 mM phosphate buffer (pH 7.4), 2 mM EDTA, 0.2 mg/mL BSA, 50 uM TRFS-red, and varying concentrations of recombinant TrxR1, the reaction is initiated by adding 200 uM NADPH at room temperature. The fluorescence increase (excitation 550 nm, emission 620 nm) is monitored in kinetic mode for 10-30 minutes. The initial linear slope of the fluorescence increase is directly proportional to the concentration and activity of TrxR, allowing for the determination of the probe's response kinetics and sensitivity.
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| Cell Assay |
For in vitro cell imaging, HeLa or other adherent cells are seeded on glass-bottom culture dishes or 96-well plates and allowed to adhere overnight. The culture medium is removed, and the cells are incubated with serum-free medium containing 1 microM TRFS-red for 30-120 minutes at 37degC in a CO2 incubator [3L38-L39]. For inhibition studies, cells may be pre-treated with an inhibitor like auranofin (1 microM) for 60 minutes. After incubation, the cells are washed twice with PBS to remove excess probe. The cells are then imaged using a fluorescence microscope or a high-content imaging system with a TRITC/Cy3 filter set. Fluorescence intensity can be quantified to measure relative TrxR activity.
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| Animal Protocol |
TRFS-red can be used for in vivo imaging of TrxR activity in small animal models. A typical protocol involves the subcutaneous or intravenous (i.v.) administration of TRFS-red (e.g., in 100 microL of PBS containing 5% DMSO) into mice at a dose of 0.5-2 mg/kg. The animal is then anesthetized with isoflurane, and whole-body fluorescence imaging is performed using an in vivo imaging system (IVIS) with appropriate excitation and emission filters (e.g., 605 nm excitation, 660 nm emission). Images are taken at various time points (e.g., 0, 15, 30, 60, and 120 minutes) post-injection to monitor the activation of the probe in different tissues. A decrease in the fluorescent signal can be correlated with disease states where TrxR activity is altered, such as in a mouse model of stroke [25L36-L37].
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| ADME/Pharmacokinetics |
TRFS-red is a chemical probe used as a diagnostic tool, not a therapeutic drug; thus, it is not intended to have traditional pharmacokinetic properties. Its behavior in vivo is characterized by its distribution and clearance, which is important for imaging applications. The red-shifted emission of TRFS-red is critical for its in vivo application, as it allows for deeper tissue penetration and minimizes autofluorescence from the animal's body, providing a higher signal-to-noise ratio for imaging TrxR activity in live animals. The probe can be detected in tissues such as the brain and is cleared over time as the fluorophore is released.
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| Toxicity/Toxicokinetics |
TRFS-red is a fluorescent probe designed for research applications, not a therapeutic drug, so formal toxicity studies (e.g., LD50) are generally not conducted. As a research chemical, it should be handled with standard laboratory safety precautions. The compound has been used successfully for live cell and in vivo imaging, indicating that at the effective doses (e.g., 1 microM in vitro, 1-2 mg/kg in vivo) and exposure times used, it is generally non-cytotoxic and well-tolerated. However, the effects of the released fluorophore are not fully characterized, so caution is always advised. It is strictly for research use only and not for human consumption.
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| References | |
| Additional Infomation |
TRFS-red is the first red-emission off-on probe for thioredoxin reductase (TrxR) [3L24-L25]. It was developed as a chemical probe for research into the dynamics of redox processes within cells, particularly in the context of oxidative stress and disease. It is valuable for the visualization of enzyme activity linked to signaling pathways that regulate cell growth and apoptosis, especially in cancer research [3L26-L29]. The probe, with its improved spectral properties, is an important tool for studying the role of the TrxR system in various diseases, including cancer and neurodegenerative conditions. It is not a therapeutic drug, has not been approved for clinical use, and is not in clinical trials.
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| Molecular Formula |
C28H28CLN3O11S2
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| Molecular Weight |
682.12
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| Appearance |
Brown to black solid powder
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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: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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 :~5 mg/mL (~7.33 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.5 mg/mL (0.73 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 5.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O 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 | 1.4660 mL | 7.3301 mL | 14.6602 mL | |
| 5 mM | 0.2932 mL | 1.4660 mL | 2.9320 mL | |
| 10 mM | 0.1466 mL | 0.7330 mL | 1.4660 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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