Amplex red monoamine oxidase assay kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Amplex Red Monoamine Oxidase Assay Kit is a fluorometric assay used to measure the activity of monoamine oxidase, an enzyme involved in the metabolism of neurotransmitters. The kit provides a sensitive and specific method for detecting monoamine oxidase activity in a variety of sample types.

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Lab products found in correlation

M1000 pro, by Tecan (1 mentions) Spectramax gemini em, by Molecular Devices (1 mentions) Bca protein assay kit, by Merck Group (1 mentions) Spectramax m5, by Molecular Devices (1 mentions) Infinite m200 pro microplate reader, by Tecan (1 mentions) M7316, by Merck Group (1 mentions) Pargyline, by Thermo Fisher Scientific (1 mentions) Norepinephrine hydrochloride, by Merck Group (1 mentions) Nisoxetine hydrochloride, by Merck Group (1 mentions) Corticosterone, by Bio-Techne (1 mentions) Semicarbazide hydrochloride, by Merck Group (1 mentions) Catalase, by Merck Group (1 mentions) Pargyline hydrochloride, by Cayman Chemical (1 mentions) El808 plate reader, by Agilent Technologies (1 mentions)

Close spelling variants of this product

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20 protocols using amplex red monoamine oxidase assay kit

Quantifying LOX Family Enzyme Activity

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LOX family activity assays were performed with the Amplex Red Monoamine Oxidase Assay Kit (molecular probes, Invitrogen). For the supernatant activity, each cell lines were plated at the same density (750.000 cells per 10 cm dishes) and 48 hours after, cell mediums were collected. Measured were performed on 20 μl of medium. For tumor samples, LOX family activities were measured on 2 μg of proteins after cryogenic grinding. Each sample was used and measured according to manufacturer's instructions and as previously described in [16 (link)]. Amplex red was excited at 530 nm and emission was measured at 590 nm using Tecan M1000 PRO. Assays were run in sextuplicate and activity were reported as a mean ± S.E.M of all assays.

Le Calvé B., Griveau A., Vindrieux D., Maréchal R., Wiel C., Svrcek M., Gout J., Azzi L., Payen L., Cros J., de la Fouchardière C., Dubus P., Guitton J., Bartholin L., Bachet J.B, & Bernard D. (2016). Lysyl oxidase family activity promotes resistance of pancreatic ductal adenocarcinoma to chemotherapy by limiting the intratumoral anticancer drug distribution. Oncotarget, 7(22), 32100-32112.

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Monoamine Oxidase B Activity Assay

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MAO-B activity was determined by monoamine oxidase activity using an Amplex^® Red Monoamine Oxidase Assay Kit (Molecular Probes, Cat. #A12214, Eugene, OR, USA). Briefly, the brain homogenates were incubated with benzylamine, a MAO-B substrate, with or without 1 μM selegiline for 30 min at 37 °C. The reaction was performed with equal volume of working solution at room temperature for 20 min. Fluorescence generated by H₂O₂ through 10-acetyl-3,7-dihydroxyphenoxazine in a horseradish peroxidase-coupled reaction was measured using a SpectraMax Gemini EM microplate spectrophotometer (Molecular Devices, LLC., SomJose, CA) with detection in the 530–560 nm range for excitation and 590 nm for emission.

Choi J.Y., Yun J., Hwang C.J., Lee H.P., Kim H.D., Chun H., Park P.H., Choi D.Y., Han S.B, & Hong J.T. (2019). (E)-2-methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) Phenol Ameliorates MPTP-Induced Dopaminergic Neurodegeneration by Inhibiting the STAT3 Pathway. International Journal of Molecular Sciences, 20(11), 2632.

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Quantifying SSAO Activity in Visceral Fat

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Tissue homogenates were prepared on ice by homogenizing pieces of frozen visceral fat from human subjects in 200 μL of homogenizing buffer (50 mM HEPES, 1/1000 Igepal, 150 mM NaCl, 0.25 M sucrose, 0.5 μM AEBSF and 1 : 100 protease inhibitor solution). The samples received 30 strokes with tight-fitting pistons. Samples were then centrifuged at 1000 × g for 15 min to remove the debris, and the supernatant was collected. The protein concentration of the lysate was determined by the BCA assay.
The SSAO activity was determined fluorometrically at 37 °C by measuring hydrogen peroxide production using the Amplex Red Monoamine Oxidase Assay Kit (Molecular Probes, The Netherlands) according to manufacturer’s instructions. Briefly, tissue homogenates of 10 μg protein were pre-incubated for 30 min at 37 °C with 100 μM monoamine oxidase B inhibitor pargyline. The reaction was started by the addition of 100 μL of the reaction buffer (400 μM Amplex Red, 2 U mL⁻¹ horseradish peroxidase, 2 mM benzylamine, 50 mM sodium phosphate, pH 7.4) and the fluorescence (excitation: 530 nm, emission: 590 nm) was monitored every two minutes for 60 min at 37 °C in a microplate reader. In parallel, 1 mM semicarbazide (SSAO inhibitor) was added to the same reaction mixture as a negative (background) control. Resorufin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) was used as a standard.

Yang H., Liu C.N., Wolf R.M., Ralle M., Dev S., Pierson H., Askin F., Steele K.E., Magnuson T.H., Schweitzer M.A., Wong G.W, & Lutsenko S. (2019). Obesity is associated with copper elevation in serum and tissues. Metallomics : integrated biometal science, 11(8), 1363-1371.

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Quantifying Brain MAO-B Activity

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After centrifugation (10,000×g for 20 min at 4°C) of the brain
tissue homogenate, the pellet was used to assay MAO-B activity in brain
tissue using the Amplex Red monoamine oxidase assay kit (A-12214, Molecular
Probes, Eugene, OR, USA). In brief, a reaction mixture (500 μL)
containing Amplex Red reagent (400 μM), benzylamine (2 mM) as a
specific substrate for MAO-B, and horseradish peroxidase (2 U/mL) was
prepared. The mixtures were incubated at 23°C in 96-well plates for 1
h. Fluorescence was then measured using a fluorescence reader (Molecular
Device, USA) at excitation wavelength of 560 nm and emission wavelength of
590 nm. The MAO-B activity was recorded as μM resorufin/60 min/mg
protein. The protein content of the sample was assayed using the
bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich, St. Louis, MO,
USA).

Kim D., Kim Y.H., Ham J.S., Lee S.K, & Jang A. (2020). Pig Skin Gelatin Hydrolysates Attenuate Acetylcholine Esterase Activity and Scopolamine-induced Impairment of Memory and Learning Ability of Mice. Food Science of Animal Resources, 40(2), 183-196.

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Amplex Red Monoamine Oxidase Assay

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The MAO activity of tissue and cell lysates was determined by the Amplex Red Monoamine Oxidase Assay Kit (Molecular Probes).

Wang S.H., Tsai F.C., Lin H.H., Yu T.Y., Kuo C.H., Li H.Y, & Lin M.S. (2023). Inhibition of monoamine oxidase B reduces atherosclerosis and fatty liver in mice. Clinical Science (London, England : 1979), 137(1), 17-30.

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Quantifying Monoamine Oxidase Activity

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The brains of mice were homogenized as previously described (Naoya Aoo, 2013 (link)) and the protein concentrations of the homogenates were measured as above. Monoamine oxidase (MAO) activity was determined with the Amplex Red Monoamine Oxidase Assay Kit (A12214, Molecular Probes, OR, United States). The homogenates were mixed in equal amounts with a working solution composed of Amplex Red (200 μM), horseradish peroxidase (1 U/mL), and MAO substrate (p-tyramine or benzylamine; 1 mM) and incubated at 37°C for 1 h. MAO-B activity was detected using benzylamine for the MAO-B substrate. The fluorescence intensity was measured using a fluorescence plate reader (SpectraMax M5, Molecular Devices, United States) at an excitation wavelength of 535 nm and an emission wavelength of 590 nm.

Chen J., Guo N., Ruan Y., Mai Y., Liao W, & Feng Y. (2023). Isoniazid improves cognitive performance, clears Aβ plaques, and protects dendritic synapses in APP/PS1 transgenic mice. Frontiers in Aging Neuroscience, 15, 1105095.

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Quantifying SSAO Activity via Amplex Red Assay

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SSAO activity was determined as a result of the turnover of hydrogen peroxide, which was measured by Amplex Red Monoamine Oxidase Assay Kit (A12214, Molecular Probes, Eugene, OR, USA). Briefly, 100μg of protein was incubated at room temperature with clorgyline 1 μM (monoamine oxidase-A inhibitor), pargyline 3 μM (monoamine oxidase-B inhibitor), benzylamine 2 mM (substrate of SSAO), 100 μM Amplex Red reagent, and 1 U/ml HRP, with or without semicarbazide 1mM.
Absorbance at 570 nm was measured every 5 minutes for 30 minutes. A standard curve was plotted using different solutions with known hydrogen peroxide concentrations. Production rate of hydrogen peroxide is calculated and expressed as [H 2 O 2 ]/min/μg protein. SSAO activity was determined by the difference between the production rates of hydrogen peroxide with and without semicarbazide. The linearity (R2) of this assay is 0.9989~1.0000 and the intra-or inter-assay CV is 0.5~3.8% in our lab.

Wang S.H., Yu T.Y., Tsai F.C., Weston C.J., Lin M.S., Hung C.S., Kao H.L., Li Y.I., Solé M., Unzeta M., Chen Y.L., Chuang L.M, & Li H.Y. (2018). Inhibition of semicarbazide-sensitive amine oxidase reduces atherosclerosis in apolipoprotein E-deficient mice. Translational research : the journal of laboratory and clinical medicine, 197.

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Quantifying Monoamine Oxidase B Activity

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Enzymatic activity of MaOB was measured as described previously (Jo et al., 2014) . Frozen hippocampus tissues from each mouse were homogenized, and large debris was removed by weak centrifugation. Subsequently, the supernatant was collected, and mitochondria-rich fraction was obtained by centrifuging the supernatant at 13,000 rpm for 20 minutes. The pellet was resuspended in phosphate buffer, and 30 mg were used in each well to determine the activity of the MaOB using an Amplex Red Monoamine oxidase Assay Kit (Molecular Probes) according to the manufacturer's instructions. After enzyme reaction, hydrogen peroxide, which is produced by MaOB activity, is measured by a color change of Amplex red reagent. The color change was quantified by measuring absorbance at 590 nm with Infinite M200 PRO microplate reader (TECAN).

Roy U., Stute L., Höfling C., Hartlage-Rübsamen M., Matysik J., Roβner S, & Alia A. (2018). Sex- and age-specific modulation of brain GABA levels in a mouse model of Alzheimer's disease. Neurobiology of aging, 62.

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Measuring SSAO Activity via Amplex Red

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SSAO activity was determined as a result of the turnover of hydrogen peroxide, which was measured by the Amplex Red Monoamine Oxidase Assay Kit (Invitrogen, Beijing, China). Briefly, 100 g of protein was incubated at room temperature with clorgyline 1 M (monoamine oxidase-A inhibitor), pargyline 3 M (monoamine oxidase-B inhibitor), benzylamine 2 mM (substrate of SSAO), Amplex Red reagent 100 M, and horseradish peroxidase 1 U/mL, with or without semicarbazide 1 mM. Absorbance at 570 nm was measured every 5 min for 30 min. A standard curve was plotted using different solutions with known hydrogen peroxide concentrations. The production rate of hydrogen peroxide was calculated and expressed as [H₂O₂]/min/g protein. SSAO activity was determined by the difference between the production rates of hydrogen peroxide with and without semicarbazide.

Yan L., Sun C., Zhang Y., Zhang P., Chen Y., Deng Y., Er T., Deng Y., Wang Z, & Ma H. (2023). The Biological Implication of Semicarbazide-Sensitive Amine Oxidase (SSAO) Upregulation in Rat Systemic Inflammatory Response under Simulated Aerospace Environment. International Journal of Molecular Sciences, 24(4), 3666.

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Determination of MAO Enzyme Inhibition

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The determination of MAO-A and MAO-B inhibition was performed using commercially available recombinant human MAO-A and MAO-B enzymes expressed in baculovirus-infected insects sells (Sigma-Aldrich, M7316 and M7441) applying the commercially available Amplex® Red monoamine oxidase assay kit (Invitrogen A12214). The assays were performed as previously described. The determination of rat MAO-B inhibition was performed using mitochondrial-enriched fractions from male Sprague Dawley rat livers. The assays were conducted as previously described (Stössel et al., 2013 (link)).

Koch P., Brunschweiger A., Namasivayam V., Ullrich S., Maruca A., Lazzaretto B., Küppers P., Hinz S., Hockemeyer J., Wiese M., Heer J., Alcaro S., Kiec-Kononowicz K, & Müller C.E. (2018). Probing Substituents in the 1- and 3-Position: Tetrahydropyrazino-Annelated Water-Soluble Xanthine Derivatives as Multi-Target Drugs With Potent Adenosine Receptor Antagonistic Activity. Frontiers in Chemistry, 6, 206.

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