Bis n methylacridinium nitrate

Manufactured by Merck Group

Sourced in United States, United Kingdom

Bis-N-methylacridinium nitrate is a chemical compound used as a laboratory reagent. It is a bright yellow crystalline solid that is soluble in water and organic solvents. The compound is commonly used in various analytical and experimental procedures, but a detailed description of its core function would require further information to maintain an unbiased and factual approach.

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11 protocols using bis n methylacridinium nitrate

Myocardial Magnesium and Oxidative Stress Analysis

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Blood samples from the aorta were immediately centrifuged at 3,000 g for 10 min, and the serum was stored at -70°C until further analysis. Serum Mg²⁺ concentrations were determined using an automated chemical analyzer.
Myocardium at the border zone was processed according to the manufacturer’s instructions. The Mg²⁺ content of the myocardium was assessed by chopping the LV and digesting them with nitric acid (Sigma), followed by measurements using an atomic absorption spectrophotometer (Shimadzu, Tokyo, Japan) as described previously [29 (link)]. The total level of norepinephrine in the myocardium was measured using a commercial ELISA kit (Noradrenalin ELISA, IBL Immuno-Biological Laboratories, Hamburg, Germany). Myocardial peroxynitrite formation was estimated by measuring the level of free nitrotyrosine (as a marker for peroxynitrite formation) using an ELISA kit (Cayman Chemical, Ann Arbor, MI, USA) in myocardial homogenates.
Superoxide production at the border zone was also measured using lucigenin (5 μmol/L bis-N-methylacridinium nitrate, Sigma, St. Louis, MO) and enhanced chemiluminescence as previously described [28 (link)]. The specific chemiluminescence signal was calculated after subtraction of the background signal and expressed as counts per minute per milligram weight (cpm/mg).

Lee T.M., Chang N.C, & Lin S.Z. (2018). Effect of proton pump inhibitors on sympathetic hyperinnervation in infarcted rats: Role of magnesium. PLoS ONE, 13(8), e0202979.

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Resolvin E1 and Oxidative Stress Evaluation

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Blood samples from ascending aortas of the rats were collected at the end of the study, and serum was separated by centrifugation. To assure EPA levels more relevant to human populations, serum Resolvin E1 levels were measured by using a commercially available kit (MyBiosource, Inc) and following the manufacturer's instructions.
Myocardial production of superoxide in the remote zone was evaluated with lucigenin (5 µmol/L bis‐N‐methylacridinium nitrate, Sigma)‐enhanced chemiluminescence as reported in a previous study.²⁰ The chemiluminescent signal was calculated by subtracting background activity and was expressed as counts/min/mg weight (cpm/mg).
Myocardial peroxynitrite formation was estimated by measuring the level of free nitrotyrosine (as a marker for peroxynitrite formation) by ELISA (Cayman Chemical) in myocardial homogenates.

Chen W., Chen S., Wu D., Lee C, & Lee T. (2020). Effect of icosapent ethyl on susceptibility to ventricular arrhythmias in postinfarcted rat hearts: Role of GPR120‐mediated connexin43 phosphorylation. Journal of Cellular and Molecular Medicine, 24(16), 9267-9279.

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Cardiac Biomarker Quantification Protocol

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Blood samples were separated by centrifugation at 3000g for 10 min at 4°C. E2 levels were measured using a commercial kit (Sigma, St. Louis, MO).
Lucigenin‐enhanced chemiluminescence (5 μM, bis‐N‐methylacridinium nitrate, Sigma, St. Louis, MO) was used to measure myocardial superoxide production from the border zone as previously described.²⁸ Specific chemiluminescence signals were obtained by subtracting background activity and expressed as counts per minute per milligram weight (cpm/mg).
Myocardial IL‐10 activity for M2c was assayed from the border zones, and the myocardial membrane‐bound IL‐10 fraction was evaluated using a commercially available ELISA kits (R&D Systems). Norepinephrine levels of the supernatant proteins from the remote zone were also evaluated using a commercially available ELISA kit (Noradrenalin ELISA, IBL Immuno‐Biological Laboratories Co., Hamburg, Germany).

Lee C., Chen S, & Lee T. (2022). 17β‐Oestradiol facilitates M2 macrophage skewing and ameliorates arrhythmias in ovariectomized female infarcted rats. Journal of Cellular and Molecular Medicine, 26(12), 3396-3409.

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Preparation of Chemical Reagents

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Tunicamycin, ACh, SNP, phenylephrine, Tween-20, bis-N-methylacridinium nitrate, DETCA, DPI, β-NADPH, Tris-base were purchased from Sigma-Aldrich (St. Louis, MO, USA). DiOHF was purchased from Indofine and dissolved in DMSO. TUDCA was purchased from Calbiochem. Bovine serum albumin (BSA) was purchased from Santa Cruz (Dallas, Texas, USA). Kreb’s salts were purchased from BDH Limited and BDH Laboratory Supplies (Poole, UK).

Lau Y.S., Mustafa M.R., Choy K.W., Chan S.M., Potocnik S., Herbert T.P, & Woodman O.L. (2018). 3′,4′-dihydroxyflavonol ameliorates endoplasmic reticulum stress-induced apoptosis and endothelial dysfunction in mice. Scientific Reports, 8, 1818.

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Measuring Retinal Superoxide Production

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Subgroups of dark-adapted B6 and S6 mice were treated with SI. These mice were maintained in darkness for at least 16 hours before euthanasia and retina removal. Superoxide production was measured on each retina using a standard lucigenin (bis-N-methylacridinium nitrate; Sigma-Aldrich Corp.).^{23 (link)}

Berkowitz B.A., Podolsky R.H., Lenning J., Khetarpal N., Tran C., Wu J.Y., Berri A.M., Dernay K., Shafie-Khorassani F, & Roberts R. (2017). Sodium Iodate Produces a Strain-Dependent Retinal Oxidative Stress Response Measured In Vivo Using QUEST MRI. Investigative Ophthalmology & Visual Science, 58(7), 3286-3293.

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Superoxide Production in Retina of Dark-Reared Mice

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Subgroups of dark-reared P23 wt and Pde6b^rd10 mice were maintained in darkness for at least 16 hours before euthanasia, and their retinas were removed in a dim room. Superoxide production was measured on one retina by using a standard lucigenin (bis-N-methylacridinium nitrate; Sigma-Aldrich Corp.) assay in which each retina preparation was given three carefully timed and gentle shakes, followed each time with three measurements (total of nine measurements).^{13 (link)} Data from one male Pde6b^rd10 mouse were not used because the laboratory lights were accidentally turned on. Because each subgroup had data collected on different days, companion measurements were obtained each day from 3 dark-adapted 2-month-old wt mice. This allowed us to normalize that day's data to the 2-month-old wt mice and compare data from each group across days.

Berkowitz B.A., Podolsky R.H., Berri A.M., Dernay K., Graffice E., Shafie-Khorassani F, & Roberts R. (2018). Dark Rearing Does Not Prevent Rod Oxidative Stress In Vivo in Pde6brd10 Mice. Investigative Ophthalmology & Visual Science, 59(3), 1659-1665.

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Evaluating Myocardial Inflammation and Oxidative Stress

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Serum IL‐1β levels at day 3 after MI were assessed using ELISA kits (R&D Systems, Minneapolis, MN, USA) and expressed as pg/mL.
Collagen histology was confirmed by hydroxyproline assay using a method adapted from that reported by Stegemann and Stalder.²⁵ Remote zone samples were immediately frozen in liquid nitrogen and stored at −80°C until the hydroxyproline content per weight of tissue was measured.
Myocardial superoxide production at the remote zone was measured using lucigenin (5 μmol/L bis‐N‐methylacridinium nitrate, Sigma) enhanced chemiluminescence as previously described.²³ The chemiluminescence in the sample was continuously measured for a total of 300 seconds. The specific chemiluminescence signal was calculated after subtracting background activity and was expressed as counts/min/mg weight (cpm/mg).

Lee T., Harn H., Chiou T., Chuang M., Chen C., Chuang C., Lin P, & Lin S. (2020). Host pre‐conditioning improves human adipose–derived stem cell transplantation in ageing rats after myocardial infarction: Role of NLRP3 inflammasome. Journal of Cellular and Molecular Medicine, 24(21), 12272-12284.

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Measuring Retinal Superoxide in Mice

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Subgroups of dark-adapted B6 and S6 mice were treated with d-cis-diltiazem approximately 4 hours before enucleation. These mice were maintained in darkness for at least 16 hours before euthanasia and retina removal. Superoxide production was measured on each retina using a standard lucigenin assay (bis-N-methylacridinium nitrate; Sigma-Aldrich Corp.).^{43 (link)}

Berkowitz B.A., Podolsky R.H., Farrell B., Lee H., Trepanier C., Berri A.M., Dernay K., Graffice E., Shafie-Khorassani F., Kern T.S, & Roberts R. (2018). D-cis-Diltiazem Can Produce Oxidative Stress in Healthy Depolarized Rods In Vivo. Investigative Ophthalmology & Visual Science, 59(7), 2999-3010.

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Chemiluminescent ROS Detection Assay

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The chemiluminescent probe, Bis-N-methylacridinium nitrate (lucigenin, catalogue no. 2315-97-1); the reagents L-NAME (N5751), Rotenone (R8875), Oxypurinol (O6881), Diphenyleneiodonium chloride (DPI, D2926), Triton X-100 (T8787) and Tiron (D7389); fluorescein isothiocyanate (FITC)-conjugated wheat germ agglutinin (Lectin, L4895) and; fluorochrome-conjugated anti-goat (C2821) or anti-rabbit (C2306) secondary antibodies were purchased from Sigma-Aldrich, Dorset, UK and gp91 ds-tat (DS-TAT, AS-63818) was bought from AnaSpec, Fremont, USA. NADPH (AC328742500) was purchased from Thermo Fisher Scientific, Waltham, MA, USA. Dihydroethidium (DHE, D11347) was from Invitrogen, Paisley, UK. Polyclonal antibodies targeting Nox2 (sc-20782), Nox4 (sc-30141), phospho-Akt1/2/3 (sc-16646-R), total Akt1/2/3 (sc-8312), total ERK1/2 (sc-292838), total-p38MAPK (sc-535), α-tubulin (sc-5546) and the monoclonal antibody targeting phospho-p38MAPK (sc-166182) were from Santa Cruz Biotechnology, Dallas, TX, USA. Phospho-ERK1/2 (#9101) was purchased from Cell Signaling Technology, Danvers, MA, USA.

Bhatti S.N, & Li J.M. (2020). Nox2 dependent redox-regulation of Akt and ERK1/2 to promote left ventricular hypertrophy in dietary obesity of mice. Biochemical and biophysical research communications, 528(3).

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Quantifying Neutrophil Oxidative Burst

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Lucigenin-dependent chemiluminescence assay was performed as previously described (14 ). Briefly, 1 x 10⁶ neutrophils were added to 125 µg lucigenin (bis-N-methylacridinium nitrate, Sigma-Aldrich) in 500 µL HBSS. Chemiluminescence was measured using a LB 953 Autolumat Plus luminometer (Berthold Technologies, Bad Wildbad, Germany), and peak fluorescence recorded. Data is expressed as mean relative luminescence units (RLU).

Small A.G., Perveen K., Putty T., Patel N., Quinn P., Wechalekar M.D., Hii C.S., Quach A, & Ferrante A. (2022). Neutrophils Require Activation to Express Functional Cell-Surface Complement Receptor Immunoglobulin. Frontiers in Immunology, 13, 840510.

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