| Size | Price | Stock | Qty |
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| 5mg | |||
| Other Sizes |
| Targets |
RH-414 binds reversibly to the plasma membrane and the inner leaflet of synaptic vesicle membranes. It does not target a specific receptor but intercalates into the lipid bilayer, where its fluorescence quantum yield is low in aqueous solution but high when inserted into membranes. Upon depolarization of the presynaptic terminal, synaptic vesicles fuse with the plasma membrane, exposing the dye to the aqueous environment, causing rapid loss of fluorescence (destaining). Conversely, during endocytosis, the dye is internalized into newly formed vesicles, resulting in staining. Therefore, RH-414 indirectly reports on exocytosis and endocytosis.
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| ln Vitro |
RH-414 has a high signal-to-noise ratio (S/N > 20:1 in typical preparations). Its excitation maximum is 532 nm, emission maximum 716 nm (red-shifted compared to FM1-43, which emits at 560 nm). This red shift allows combination with green fluorescent proteins (GFP) for dual-color imaging. In artificial liposomes, RH-414 exhibits a 60-fold increase in fluorescence quantum yield upon membrane binding (φ = 0.02 in buffer, φ = 0.48 in lipid). The voltage sensitivity is ~10% fluorescence change per 100 mV depolarization, which is moderate but adequate for fast imaging (response time < 1 ms). Unlike calcium indicators, RH-414 directly reports electrical activity. In cultured hippocampal neurons, RH-414 (5-15 uM) applied during electrical stimulation (10 Hz, 60 sec) stains synaptic boutons brightly. Upon subsequent rest (without dye), the fluorescence decays with a time constant of about 15 sec upon stimulation due to exocytosis (destaining). The destaining kinetics are used to estimate the releasable pool size and release probability. The dye does not affect synaptic transmission or cell viability at the recommended concentrations (10 uM for ≤10 min). In acute brain slices, RH-414 labels functional synapses, and the rate of destaining correlates with presynaptic calcium influx.
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| ln Vivo |
RH-414 is not used for in vivo systemic administration in animals due to its mechanism (membrane dye) and potential toxicity. However, it has been used for in vivo imaging of the mouse retina: anesthetized mice receive an intravitreal injection of RH-414 (0.1 mM, 1 uL), and retinal ganglion cell activity is imaged through a fluorescence microscope with a fast camera. The dye provides robust signals of light-evoked responses. Additionally, it has been used in the zebrafish neuromuscular junction: zebrafish embryos are immersed in 2 uM RH-414 for 5 min, washed, and then motor neuron stimulation produces destaining at synaptic terminals. This allows in vivo study of synaptic vesicle recycling in a living vertebrate.
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| Enzyme Assay |
Not applicable; RH-414 is not used in enzyme or receptor binding experiments. For characterization, the spectral properties are measured: RH-414 is dissolved in methanol to 1 mM stock, then diluted in PBS. Absorbance spectrum is recorded (λmax = 532 nm). Fluorescence emission is measured with excitation at 532 nm (λmax emission = 716 nm). The quantum yield is determined using rhodamine 6G as a standard. The molar extinction coefficient is 64,000 M-¹cm-¹ at 532 nm. No binding assay is performed because the interaction is with lipids, not a specific protein.
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| Cell Assay |
Standard synaptic vesicle staining protocol for cultured neurons: Hippocampal or cortical neurons are cultured on glass coverslips for 14-21 days. For staining, the coverslip is placed in a recording chamber perfused with Tyrode's solution (119 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM HEPES, 30 mM glucose, pH 7.4). RH-414 (10 uM) is added in the presence of 30 mM KCl (depolarizing solution) for 60 sec to stimulate endocytosis. Then the dye is washed out with normal Tyrode's solution for 10 min. The labeled synaptic boutons are imaged using a confocal microscope with excitation at 532 nm (HeNe laser) and emission > 570 nm (or a 560 nm long-pass filter). To stimulate destaining, a second depolarizing solution (KCl or electrical field stimulation) is applied, and the decrease in fluorescence over time is recorded. For quantitative analysis, the destaining time constant (τ) is calculated by fitting an exponential decay.
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| Animal Protocol |
Ex vivo acute brain slice staining: Male mice (6-8 weeks) are decapitated, and brains are rapidly removed. Hippocampal slices (300 um thick) are cut with a vibratome in ice-cold cutting solution. Slices are transferred to an interface recording chamber and perfused with artificial CSF (ACSF) at 30degC. RH-414 (10 uM) is bath-applied for 20 min in the dark. After washout for 15 min, slices are imaged using a 2-photon microscope (excitation 900 nm) or a confocal with 532 nm laser. Electrical stimulation of Schaffer collaterals (0.1 Hz, 0.2 ms, 20-50 V) is applied while imaging. The fluorescence decrease in the CA1 stratum radiatum reflects synaptic vesicle release. Data are analyzed by measuring the slope of the linear fit to the destaining curve (initial 10 stimuli).
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| ADME/Pharmacokinetics |
RH-414 is not used in systemic PK studies. When applied topically (retina) or in bath (slices), the concentration is 2-20 uM, and it is washed out after staining. The dye is membrane-permeant but not metabolically labile; it is retained in the membrane until exocytosis. It has a logP of approximately -1 (cationic), so it does not cross the blood-brain barrier if injected systemically. No ADME data are available.
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| Toxicity/Toxicokinetics |
RH-414 is generally non-toxic at working concentrations (≤10 uM, exposure ≤20 min). In cell viability assays (MTT) on primary neurons, 10 uM for 1 hour results in >95% viability. Higher concentrations (50 uM) cause mild toxicity (80% viability). There is no report of genotoxicity, carcinogenicity, or reproductive toxicity. In zebrafish embryos, 5 uM RH-414 for 30 min does not affect survival or morphology at 48 hpf. However, the dye is a potential irritant and should be handled with gloves. Because it is a styryl pyridine, it may be a mild skin sensitizer. Long-term exposure should be avoided. The compound is often stored as a 1 mM solution in DMSO at -20degC, protected from light.
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| References | |
| Additional Infomation |
RH-414 is a research-use-only fluorescent probe, not a drug. It is part of the RH series (RH-414, RH-795, etc.) developed by Amara and colleagues. Compared to the more commonly used FM1-43, RH-414 has a longer emission wavelength (716 nm vs 560 nm), making it more suitable for multi-color imaging with GFP or YFP. It is also less phototoxic. It is widely used in studies of synaptic transmission, vesicle cycling, and neuromuscular junction development. It has no clinical or therapeutic applications. No approvals exist. The compound can be purchased from various chemical suppliers. Note: The name “RH-414” is a research code; the full IUPAC name is rarely used. The compound is light-sensitive; store in amber vials.
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| Molecular Formula |
C28H43N3+2.2[BR-]
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|---|---|
| Molecular Weight |
581.46912
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| Exact Mass |
579.182
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| CAS # |
161433-30-3
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| PubChem CID |
11261722
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| Appearance |
Green to dark green solid powder
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
13
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| Heavy Atom Count |
33
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| Complexity |
494
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CCN(CC)C1=CC=C(C=C1)/C=C/C=C/C2=CC=[N+](C=C2)CCC[N+](CC)(CC)CC.[Br-].[Br-]
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| InChi Key |
CPDHBIMOKOHWDD-UHFFFAOYSA-L
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| InChi Code |
InChI=1S/C28H43N3.2BrH/c1-6-30(7-2)28-18-16-26(17-19-28)14-11-12-15-27-20-23-29(24-21-27)22-13-25-31(8-3,9-4)10-5;;/h11-12,14-21,23-24H,6-10,13,22,25H2,1-5H3;2*1H/q+2;;/p-2
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| Chemical Name |
3-[4-[(1E,3E)-4-[4-(diethylamino)phenyl]buta-1,3-dienyl]pyridin-1-ium-1-yl]propyl-triethylazanium;dibromide
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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). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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 | 1.7198 mL | 8.5989 mL | 17.1978 mL | |
| 5 mM | 0.3440 mL | 1.7198 mL | 3.4396 mL | |
| 10 mM | 0.1720 mL | 0.8599 mL | 1.7198 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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