Lucigenin

The NADPH oxidase (Nox) activity inlung cancer cells was evaluated by the lucigenin chemiluminescence assay as described previously [58 (link), 59 (link)]. Following treatment, the cell membrane fraction was collected, NADPH (Sigma) (1 μM) and lucigenin (Sigma) (20 μM) were added, and chemiluminescence was determined using a Fluoroskan Ascent FL (Thermo^®) in an out-of-coincidence mode.

L-NA hydrochloride, ACh chloride, diethylamine NONOate diethylammonium salt, CGRP (8–37), TTX, L-NAME hydrochloride, 7-NI, 1400W, phentolamine, apocinin, allopurinol, lucigenin, tiron, tempol and DAF-2 (Sigma-Aldrich, Madrid, Spain) were used. Stock solutions (10 mmol/L) of drugs were made in distilled water, except for NA, which was dissolved in a NaCl (0.9%)-ascorbic acid (0.01% w/v) solution. These solutions were kept at -20°C and appropriate dilutions were made in KHS on the day of the experiment.

For superoxide anion radical assay, microorganisms were grown overnight (18 hr) in the basic mineral salt medium complemented of 0.5% of yeast extract and 0.5% of tryptone. Suspension of microorganism was triply washed and diluted with basic mineral salt medium to the concentration of 1 × 10⁸ cells per ml. Hundred microliters of culture suspension, 80 μl of basic mineral salt medium, 10 μl of 4 mM deionized water solution of lucigenin (Sigma‐Aldrich, USA), and 10 μl of the hydrocarbon were added to each well of 96‐well microplate COSTAR 3632 (USA).The control sample contained 100 μl of the suspension culture, 90 μl of basic mineral salt medium with the addition of 1% of glucose and 10 μl of 4 mM solution of lucigenin in deionized water.
The plate was incubated for 24 hr in the Luminoskan Ascent microplate luminometer (Thermo Scientific, USA) at 30°C with simultaneous chemiluminescence (CL) measurement every 30 min (48 measurements in total) (Sazykin et al., 2016, 2018). Three independent experiments were carried out and repeated 8 times.

Hepatic levels of O₂^∙− were measured using the chemiluminescence method [26 (link)]. Firstly, the weighed liver tissues of mice were homogenized in lysis buffer, pH 7.4, containing 10 mM EDTA as well as 20 mM HEPES. The samples were centrifuged for 10 min at 1000g, and, then, the aliquot of samples was incubated with a Krebs-HEPES buffer, pH 7.4, containing 5 mM lucigenin (Sigma, Shanghai, China) about 2 min at 37°C. Next, light emission data were obtained on a M200 PRO multifunctional microplate reader (TECAN, Switzerland), and the results were showed as mean light unit (MLU) min/mg protein. Levels of O₂^∙− were measured by adding SOD (350 U/mL) to the medium according to the manufacturer's instruction (R&D Systems, Minneapolis, MN, USA). In addition, liver tissues were homogenized in normal saline, and the samples were treated with equal volume of cold methanol for 60 min in a 4°C icebox. Then, the samples were centrifuged for half an hour at 10000g and we obtained the supernatant for H₂O₂ evaluation using the biochemical kits from the R&D Systems (Minneapolis, MN, USA). Protein concentration was measured using the Bradford method, and BSA was employed as the standard.