NBD-Pen
Based on 1 Customer Validation
NBD-Pen is the first fluorescence probe for lipid radicals with high selectivity and sensitivity (λex: 470 nm, λem: 530 nm). NBD-Pen specifically detects lipid derived radicals over other reactive species present in biological systems, including H2O2, ClO-, O2-?, and ?OH. NBD-Pen directly detects lipid radicals in living cells by turn-on fluorescence. NBD-Pen decreases inflammation, apoptosis, and oxidative stress markers. NBD-Pen can be studied in various disease models such as hepatic carcinoma.
For research use only. We do not sell to patients.
- Purity : 98.16%
- CAS No.: 1955505-54-0
- Formula: C19H28N5O4*
- Molecular Weight:390.46
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Storage:
4°C, protect from light
* In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)
To place orders, for customer services and technical support, please contact: MedChemExpress USA
Tel: 609-228-6898 E-mail: [email protected] [email protected]
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Biological Activity
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Chemical Information
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Solvent & Solubility
- Protocols
- Purity & Documentation
- References
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Help & FAQs
Biological Activity
Description
In Vitro
Guide (The following is our recommend protocol. This protocol is only a guide and should be modified according to your specific needs).
1. Preparation of stock solution
Use anhydrous DMSO to prepare a 10 mM NBD-Pen stock solution.
2. Preparation of working solution
Use preheated serum-free cell culture medium or PBS to dilute the stock solution to prepare a 10 μM NBD-Pen working solution.
Note: Please adjust the concentration of NBD-Pen working solution according to actual conditions and prepare it before use.
3. Spectroscopic methods
3.1 Use 100 mM phosphate buffer and 0.5% ethanol to prepare a lipid (500 μM) emulsion.
3.2 Mix this emulsion with 5 μM NBD-Pen in phosphate buffer containing 0.5% acetonitrile.
3.3 Add the LOX into the mixture at indicated concentration in phosphate buffer and incubate.
3.4 Perform fluorescence emission and ESR measurements.
3.5 Measure fluorescence spectra of the probes at excitation and emission wavelengths of 470 and 530 nm.
3.6 Monitor ESR spectrometer using a X-band (9.45 GHz) ESR spectrometer.
4. Preparation and staining of cells
4.1 Discard cell culture medium and rinse cells three times with PBS and phenol red free culture media with 10% FBS.
4.2 Add 1 μM of NBD-Pen and incubate for 10 min.
4.3 Add 30 mM of Diethylnitrosamine (DEN).
4.4 Perform fluorescence imaging immediately at 37°C under a humidified atmosphere of 5% CO2 in air.
4.5 Administer Hoechst33342 (1 μM) and SKF525A (50 μM) and incubate for 1 h, then wash with PBS.
4.6 Conduct fluorescence imaging with a confocal laser-scanning microscope with 63x or 40x objective lens.
4.7 Use the following detection lasers: Hoechst33342, λex = 405 nm, λem = 410-505 nm; NBD-Pen, λex = 458 nm, λem = 490-674 nm.
NBD-Pen (1 μM, 20 min) shows low background fluorescence in Hepal-6 cells[1].
NBD-Pen (1 μM, 10 min, followed by 30 mM DEN for another 20 min) enables visualization of intracellular lipid radicals produced by DEN activation in Hepa1-6 cells[1].
NBD-Pen (10 μM, 0-60 min) induces increased fluorescent intensity in a concentration-dependent manner in LDL with the addition of hemin (HY-19424) (0-3μM)[2].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only. Further protocols information, click here.
In Vivo
NBD-Pen (2 mM, i.v., 1-24 h after a 1 g/kg iron dectran administration) significantly increases the fluorescence intensity in LDL at 1 h after iron administration to mice with the peak at 2 h in iron overloaded model mice[2].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
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Animal Model:Rats pre-treated with SKF-525A (i.p.)[1]
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Dosage:2 mM
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Administration:Intravenous injection (i.v.) 1 h after DEN administrations
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Result:Indicated that NBD-Pen had favorable properties, including stability, a turn-on switching function and the ability to detect lipid radicals in living animals.
Chemical Information
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CAS No. 1955505-54-0
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Appearance Solid
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Molecular Weight 390.46
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Formula C19H28N5O4*
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Color Orange to red
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SMILES
[O]N1C(C)(C)CC(NC2=CC=C([N+]([O-])=O)C3=NON=C32)CC1(C)CCCCC
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Shipping
Room temperature in continental US; may vary elsewhere.
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Storage
4°C, protect from light
* In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)
Solvent & Solubility
In Vitro:
DMSO : 100 mg/mL (256.11 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
Concentration (start) × Volume (start) = Concentration (final) × Volume (final)
Protocols
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Apoptosis
Apoptosis, also called programmed cell death, is generally characterized by distinct morphological characteristics.
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TUNEL staining for apoptotic DNA fragmentation
TUNEL staining detects DNA strand breaks by using terminal deoxynucleotidyl transferase to add labeled nucleotides to exposed 3′-OH DNA termini, generating either microscopic staining in fixed cells or tissue sections, or fluorescence/cytometric signal in cell suspensions. TUNEL positivity reflects DNA fragmentation but should not be interpreted alone as definitive apoptosis, because TUNEL can also label necrotic, autolytic, mechanically damaged, or DNA-repair-associated DNA breaks.
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Annexin V plus membrane-impermeant dye apoptosis staining
Annexin V-based apoptosis assays rely on the detection of phosphatidylserine (PS) externalization from the inner leaflet of the plasma membrane to the outer leaflet, an early biochemical hallmark of apoptosis. Fluorescently labeled Annexin V binds PS in a calcium-dependent manner, enabling identification of early apoptotic cells by flow cytometry or fluorescence microscopy. When combined with a membrane-impermeant DNA-binding dye (e. g. , propidium iodide), this approach allows discrimination between viable (Annexin V−/dye−), early apoptotic (Annexin V+/dye−), and late apoptotic or necrotic (Annexin V+/dye+) cell populations by assessing membrane integrity and PS exposure.
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ROS/oxidative-stress fluorescent staining
ROS/oxidative-stress fluorescent staining uses cell-permeant fluorogenic probes that become fluorescent after oxidation inside cells or tissues; commonly used examples include DCFH-DA/DCFDA for broad cellular oxidant detection, DHE for superoxide-related signal detection, MitoSOX for mitochondrial superoxide-related signal detection, and CellROX probes for oxidative-stress-associated fluorescence readouts. The assay detects probe oxidation rather than a single ROS species unless the probe and analysis method have been chemically validated for that species. DCFH-DA enters cells, is deacetylated by intracellular esterases to DCFH, and produces fluorescent DCF after oxidation, so the readout is used as an operational measure of total cellular oxidative stress rather than a species-specific ROS measurement. DHE and MitoSOX can report superoxide-related oxidation, but red fluorescence alone can include non-specific ethidium-like oxidation products; HPLC or optimized spectral approaches are
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Apoptosis Solutions
Apoptosis is a regulated, generally non-lytic cell-death pathway that removes unwanted, damaged, infected, or abnormal cells through coordinated morphological changes, caspase activation, DNA fragmentation, and membrane remodeling. The intrinsic apoptosis pathway is controlled mainly by mitochondrial outer membrane permeabilization, BCL-2 family proteins, cytochrome c release, apoptosome formation, caspase-9 activation, and downstream executioner caspase-3/7 activation. The extrinsic apoptosis pathway is initiated by death receptors such as Fas, TNFR, and TRAIL receptors, which recruit adaptor proteins and activate caspase-8 before engaging executioner caspases or mitochondrial amplification through BID cleavage. Apoptosis is linked to many phenotypes, including cancer cell killing, tissue homeostasis, immune regulation, neurodegeneration, infection response, and treatment-induced cytotoxicity; unresolved questions include how apoptosis interacts with necroptosis, pyroptosis, ferroptos
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Research Protocol for Inflammation-related Diseases
The NLRP3 inflammasome is a cytosolic innate immune signaling platform that integrates priming signals and danger-signal activation to promote caspase-1 activation, maturation of IL-1β and IL-18, and gasdermin D-mediated pyroptotic cell death. The core experimental logic is to determine whether inflammatory disease phenotypes are driven by increased NLRP3 expression, ASC-containing inflammasome assembly, caspase-1 cleavage, GSDMD cleavage, and extracellular release of IL-1β/IL-18 rather than by nonspecific cell injury alone. The pathway is strongly linked to inflammation-related disease phenotypes because monosodium urate crystals activate NALP3/NLRP3 inflammasome signaling in gout-like crystal inflammation, cholesterol crystals activate NLRP3 inflammasomes in atherogenesis models, and DSS-induced intestinal inflammation has been reported to involve NLRP3 inflammasome activity. However, experimental colitis studies also show context-dependent protective effects of NLRP3 inflammasome co
Purity & Documentation
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Data Sheet (277 KB)
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SDS (252 KB)
- English - EN (252 KB)
- Français - FR (252 KB)
- Deutsch - DE (252 KB)
- Norwegian - NO (252 KB)
- Español - ES (252 KB)
- Swedish - SV (252 KB)
- Italian - IT (252 KB)
- Korean - KR (252 KB)
- Portuguese - PT (252 KB)
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Handling Instructions (2659 KB)
References
[1]. Yamada, K., et al., (2016). Fluorescence probes to detect lipid-derived radicals. Nature chemical biology, 12(8), 608–613. [Content Brief]
[2]. Ishida Y, et al. Detection and inhibition of lipid-derived radicals in low-density lipoprotein. Free Radic Biol Med. 2017 Dec;113:487-493. [Content Brief]
Complete Stock Solution Preparation Table
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
| Optional Solvent | Concentration Solvent Mass | 1 mg | 5 mg | 10 mg | 25 mg |
|---|---|---|---|---|---|
| DMSO | 1 mM | 2.5611 mL | 12.8054 mL | 25.6108 mL | 64.0270 mL |
| 5 mM | 0.5122 mL | 2.5611 mL | 5.1222 mL | 12.8054 mL | |
| 10 mM | 0.2561 mL | 1.2805 mL | 2.5611 mL | 6.4027 mL | |
| 15 mM | 0.1707 mL | 0.8537 mL | 1.7074 mL | 4.2685 mL | |
| 20 mM | 0.1281 mL | 0.6403 mL | 1.2805 mL | 3.2014 mL | |
| 25 mM | 0.1024 mL | 0.5122 mL | 1.0244 mL | 2.5611 mL | |
| 30 mM | 0.0854 mL | 0.4268 mL | 0.8537 mL | 2.1342 mL | |
| 40 mM | 0.0640 mL | 0.3201 mL | 0.6403 mL | 1.6007 mL | |
| 50 mM | 0.0512 mL | 0.2561 mL | 0.5122 mL | 1.2805 mL | |
| 60 mM | 0.0427 mL | 0.2134 mL | 0.4268 mL | 1.0671 mL | |
| 80 mM | 0.0320 mL | 0.1601 mL | 0.3201 mL | 0.8003 mL | |
| 100 mM | 0.0256 mL | 0.1281 mL | 0.2561 mL | 0.6403 mL |
NBD-Pen Related Classifications
HY-129109 Related Classifications
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