NBD-Pen

This protocol is for reference only and should be modified according to your specific requirements.

I. NBD-Pen is used for detecting lipid free radicals in the liver of rats induced by DEN.

1. A single dose of diethylnitrosamine (DEN) at 100mg per kg body weight, was injected intraperitoneally into 8-wk-old F344male rats.

2. After DEN administration (1, 6 or 24h) 8-wk-old F344 male rats were anesthetized with a solution of hydrochloric acid medetomidine, midazolam, and butorphanol.

3. The anesthetized rats then received intravenous injections of 2mM NBD-Pen (1ml/kg body weight) in 50% polyethylene glycol 300(PEG300).

4. NBD-Pen fluorescence measurement.

4.1. After euthanasia using an over dose of anesthesia, the liver was removed and tissue samples were homogenized with a Potter-type homogenizer in three volumes (w/v) of chilled 1.15% KCl solution.

4.2. Protein content in the homogenized liver and serum samples was determined using a BCA Protein Assay kit (Cat.No.: GK10009).

4.3. Lipid solutions extracted from liver and serum samples according to the method of Bligh & Dyer^[2] were measured for fluorescence intensity of NBD-Pen (λex/λem = 470/530nm) by a fluorescent microplate reader.

5. Fluorescence imaging of liver sections.

5.1 Frozen tissue sections from the rat livers were mounted on slides in DAPI Fluoromount-G Antifade Mounting Medium (Cat.No.: GB30154).

5.2. The fluorescence of NBD-Pen in each section was visualized using BIOREVO BZ-9000 with GFP-BP filter (λex = 470/40 nm, λem = 535/50nm).

II. NBD-Pen is used for detecting lipid free radicals in cells.

1.1 Preparation of stock solution

Use anhydrous DMSO to prepare a 10mM 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 10μM 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 100mM phosphate buffer and 0.5% ethanol to prepare a lipid (500μM) emulsion.

2.2 Mix this emulsion with 5μM 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 530nm.

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 10min.

3.3 Add 30mM of Diethylnitrosamine (DEN).

3.4 Perform fluorescence imaging immediately at 37°C under a humidified atmosphere of 5% CO₂ in air.

3.5 Administer Hoechst33342 (1μM) and SKF525A (50μM) and incubate for 1h, 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, λex = 405 nm, λem = 410-505 nm; NBD-Pen, λex = 458 nm, λem = 490-674 nm.

References:
[1]. Yamada, Ki., Mito, F., Matsuoka, Y. et al. Fluorescence probes to detect lipid-derived radicals. Nat Chem Biol 12, 608–613 (2016).
[2]. Bligh, E.G. & Dyer, W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917 (1959).