To determine the activation of BMDNs infected with each bacterial strain separately we measured the enzymatic activity of MPO using the Neutrophil Myeloperoxidase Activity Assay Kit (Cayman Chemical). Briefly, infected BMDNs were incubated and at 1 and 3 hpi the color intensity of the 3,3′,5,5′-tetramethyl-benzidine (TMB), which is proportional to the amount of MPO in the sample and is detectable at 650 nm, obtaining the enzymatic activity, in μmoles/min./ml. To obtain the enzymatic specific activity, the results were normalized to the total protein concentration in the samples. Negative control of non-infected neutrophils and free bacteria were used for normalization, in addition to the negative controls with MPO inhibitor (4-aminobenzhydrazide) provided by the kit.
Neutrophil myeloperoxidase activity assay kit
Manufactured by Cayman Chemical
Sourced in United States
The Neutrophil Myeloperoxidase Activity Assay Kit is a laboratory product designed to measure the enzymatic activity of myeloperoxidase, an important enzyme found in neutrophils. The kit provides the necessary reagents and protocols to quantify myeloperoxidase levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Netosis assay kit, by Cayman Chemical (4 mentions)
Flexstation 3, by Molecular Devices (2 mentions)
Tetramethylbenzidine tmb, by Merck Group (1 mentions)
Neutrophil elastase activity assay kit, by Abcam (1 mentions)
Citrullinated histone h3 clone 11d3 elisa kit, by Cayman Chemical (1 mentions)
Sytox green, by Thermo Fisher Scientific (1 mentions)
Sytox orange, by Thermo Fisher Scientific (1 mentions)
9 protocols using neutrophil myeloperoxidase activity assay kit
1
Measuring Neutrophil Myeloperoxidase Activity
2
Quantification of Neutrophil MPO Activity
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Myeloperoxidase (MPO) is an enzyme that is released upon neutrophils stimulation and therefore serves as an index of neutrophil infiltration (39 (link)). MPO was quantified in BAL using the Neutrophil Myeloperoxidase Activity Assay Kit (Cayman Chemical) according to the manufacturer’s instructions. Briefly, samples were diluted in assay buffer and 3,3′,5,5′-tetramethylbenzidine (TMB), which reacts with MPO yielding a blue color detectable by its absorbance at 650 nm. Plate was incubated for 10 min at room temperature and then absorbance was read.
3
Measuring Neutrophil Extracellular Trap Enzymes
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Activity of NE in the NET samples was measured according to the instructions “Performing the Elastase Activity Assay” of the NETosis Assay Kit from Cayman Chemical (Ann Arbor, MI, USA). The absorbance was measured after 2 h at 405 nm with a microplate reader (Flex Station® 3, Molecular Devices, San Jose, CA, USA). NE activity was determined relative to a NE standard curve. For each treatment, the delta of NE activity after PMA stimulus and accumulated NE activity without PMA stimulus was displayed to eliminate the background produced by cell death or degranulation.
The activity of MPO in the NET samples was measured according to the instructions “Performing the Assay” of the Neutrophil Myeloperoxidase Activity Assay Kit from Cayman Chemical (Ann Arbor, MI, USA). We purchased 4-aminobenzhydrozide (4-ABH) and tetramethylbenzidine (TMB) from Sigma-Aldrich (Saint Louis, MO, USA). Absorbance was measured after 30 min at 650 nm using a microplate reader (Flex Station® 3, Molecular Devices, San Jose, CA, USA). MPO activity was determined relative to an MPO standard curve. For each treatment, the delta of MPO activity after PMA stimulus and accumulated MPO activity without PMA stimulus is displayed to eliminate the background due to cell death or degranulation.
The activity of MPO in the NET samples was measured according to the instructions “Performing the Assay” of the Neutrophil Myeloperoxidase Activity Assay Kit from Cayman Chemical (Ann Arbor, MI, USA). We purchased 4-aminobenzhydrozide (4-ABH) and tetramethylbenzidine (TMB) from Sigma-Aldrich (Saint Louis, MO, USA). Absorbance was measured after 30 min at 650 nm using a microplate reader (Flex Station® 3, Molecular Devices, San Jose, CA, USA). MPO activity was determined relative to an MPO standard curve. For each treatment, the delta of MPO activity after PMA stimulus and accumulated MPO activity without PMA stimulus is displayed to eliminate the background due to cell death or degranulation.
4
Neutrophil Activation and Enzyme Assays
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Isolated neutrophils were stimulated with 100 nM phorbol myristate acetate and seeded at a density of 25 × 104 cells in a 96‐well plate in the presence or absence of 20 × 103 stromal cells. Cells were cultured for 2 hours and 30 minutes before MPO and NE activities were assessed using the Neutrophil Myeloperoxidase Activity Assay kit (Cayman Chemical, Ann Arbor, Michigan) and Neutrophil Elastase Activity Assay Kit (Abcam, Cambridge, Massachusetts). MPO activity was assessed by incubating the cells supernatant with MPO substrate for 10 minutes. Absorbance was read using a plate reader at 650 nm. NE activity was assessed by incubating with NE substrate for 20 minutes and measuring the output on a fluorescent microplate reader at Ex/Em = 380/500 nm.
5
Neutrophil MPO and NET Quantification
6
Quantifying Neutrophil Activation and NETosis
7
Measurement of Myocardial Myeloperoxidase Activity
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The myocardial myeloperoxidase (MPO) activity was measured as an index of myocardial neutrophil infiltration with the Neutrophil Myeloperoxidase Activity Assay Kit (#600620, Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions.23
8
Quantifying Neutrophil Extracellular Traps
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In order to quantify NET formation, we measured MPO and NE as markers of NET formation, together with cell-free DNA.
cfDNA assay: The amount of cfDNA was measured as described elsewhere [30 (link)], with replacement of SYTOX Green with SYTOX Orange (Thermo Fisher, Waltham, MA, USA). Fluorescence was measured at 544 nm for extinction and 590 nm for emission with a cutoff at 570 nm, using a microplate reader (Flex Station 3, Molecular Devices, San Jose, CA, USA).
NE assay: The amount of NE was measured according to the instructions of the NETosis Assay Kit from Cayman Chemical (Ann Arbor, MI, USA). Absorbance was measured at 405 nm with a microplate reader (Flex Station 3, Molecular Devices, San Jose, CA, USA).
MPO assay: The amount of MPO was measured according to the instructions of the Neutrophil Myeloperoxidase Activity Assay Kit from Cayman Chemical (Ann Arbor, MI, USA). Absorbance was measured at 650 nm with a microplate reader (Flex Station 3, Molecular Devices, San Jose, CA, USA).
cfDNA assay: The amount of cfDNA was measured as described elsewhere [30 (link)], with replacement of SYTOX Green with SYTOX Orange (Thermo Fisher, Waltham, MA, USA). Fluorescence was measured at 544 nm for extinction and 590 nm for emission with a cutoff at 570 nm, using a microplate reader (Flex Station 3, Molecular Devices, San Jose, CA, USA).
NE assay: The amount of NE was measured according to the instructions of the NETosis Assay Kit from Cayman Chemical (Ann Arbor, MI, USA). Absorbance was measured at 405 nm with a microplate reader (Flex Station 3, Molecular Devices, San Jose, CA, USA).
MPO assay: The amount of MPO was measured according to the instructions of the Neutrophil Myeloperoxidase Activity Assay Kit from Cayman Chemical (Ann Arbor, MI, USA). Absorbance was measured at 650 nm with a microplate reader (Flex Station 3, Molecular Devices, San Jose, CA, USA).
9
Quantifying Neutrophil MPO Activity in Aeromonas Infection
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Comparison of MPO activity in neutrophil of control and A. salmonicida infected L. rohita was analyzed by measuring the MPO activity in the neutrophil population using the Neutrophil Myeloperoxidase activity assay kit (Cayman Chemical) (Prokopowicz et al., 2012) (link). The samples were loaded onto 96 well plate according to the kit and the blue color yielded due to reaction of 3,3',5,5'-tetramethyl benzidine (TMB) with MPO was observed at an absorbance of 650 nm using ELISA plate reader.
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