NBD-Pen 

NBD-Pen is the first fluorescence probe for lipid radicals with high selectivity and sensitivity (λ_ex: 470 nm, λ_em: 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^[1].

Guide (The following is our recommend protocol. This protocol is only a guide and should be modified according to your specific needs).
1.1 Preparation of stock solution
Use anhydrous DMSO to prepare a 10 mM NBD-Pen stock solution.
1.2 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.
2. Spectroscopic methods
2.1 Use 100 mM phosphate buffer and 0.5% ethanol to prepare a lipid (500 μM) emulsion.
2.2 Mix this emulsion with 5 μM NBD-Pen in phosphate buffer containing 0.5% acetonitrile.
2.3 Add the LOX into the mixture at indicated concentration in phosphate buffer and incubate.
2.4 Perform fluorescence emission and ESR measurements.
2.5 Measure fluorescence spectra of the probes at excitation and emission wavelengths of 470 and 530 nm.
2.6 Monitor ESR spectrometer using a X-band (9.45 GHz) ESR spectrometer.
3. Preparation and staining of cells
3.1 Discard cell culture medium and rinse cells three times with PBS and phenol red free culture media with 10% FBS.
3.2 Add 1 μM of NBD-Pen and incubate for 10 min.
3.3 Add 30 mM of DEN.
3.4 Perform fluorescence imaging immediately at 37℃ under a humidified atmosphere of 5% CO₂ in air.
3.5 Administer Hoechst33342 (1 μM) and SKF525A (50 μM) and incubate for 1 h, then wash with PBS.
3.6 Conduct fluorescence imaging with a confocal laser-scanning microscope with 63x or 40x objective lens.
3.7 Use the following detection lasers: Hoechst33342, λ_ex = 405 nm, λ_em = 410-505 nm; NBD-Pen, λ_ex = 458 nm, λ_em = 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.

NBD-Pen (2 mM, i.v., 1-24 h after DEN administration) increases fluorescence intensity 1 h after DEN administration in plasma and liver of rat models^[1].
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.

390.46

C₁₉H₂₈N₅O₄^*

1955505-54-0

Solid

Orange to red

[O]N1C(C)(C)CC(NC2=CC=C([N+]([O-])=O)C3=NON=C32)CC1(C)CCCCC

Room temperature in continental US; may vary elsewhere.

4°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

* 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.

• [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]