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Enzchek myeloperoxidase activity assay kit

Manufactured by Thermo Fisher Scientific
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The EnzChek Myeloperoxidase Activity Assay Kit is a laboratory product designed to measure the enzymatic activity of myeloperoxidase, an important enzyme involved in various biological processes. The kit provides the necessary reagents and protocols to perform this specific assay.

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Lab products found in correlation

Mouse neutrophil elastase elisa kit, by Cusabio (2 mentions) Caspase 3 colorimetric assay kit, by Abcam (1 mentions) Taqman fam labeled probes, by Thermo Fisher Scientific (1 mentions) Scion image, by Techcomp Instruments (1 mentions)

6 protocols using enzchek myeloperoxidase activity assay kit

1

Myocardial Infarction Myeloid Cell Infiltration

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A separate set of hearts were collected at 1, 2, and 3 days post-mI/R for assessment of myeloid cell infiltration of the infarcted myocardium, by MPO assay, as previously described10 (link). Briefly, animals were killed by isoflurane and cervical dislocation, and hearts were rinsed in phosphate buffered saline, weighed, and stored at −80 °C until use. MPO was isolated using the EnzChek Myeloperoxidase Activity Assay Kit (Life Technologies; Molecular Probes). Tissue was homogenized in 1 mL of 50 mM potassium phosphate buffer (pH 6.0) with 0.5% (w/v) hexadecyltrimethyl ammonium bromide. The homogenate was sonicated and freeze-thawed three times, then centrifuged (20,000 rpm, 15 min) using a Beckman Ultracentrifuge (LE-80K). The supernatant containing MPO was collected and quantified. Values were normalized to tissue weight. n = 5 hearts/group, and all samples were run in duplicate.
Reitz C.J., Alibhai F.J., Khatua T.N., Rasouli M., Bridle B.W., Burris T.P, & Martino T.A. (2019). SR9009 administered for one day after myocardial ischemia-reperfusion prevents heart failure in mice by targeting the cardiac inflammasome. Communications Biology, 2, 353.
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2

Quantifying Inflammatory Markers in Lung Samples

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Caspase-3 activity in lung and cell lysates was assayed using a Caspase-3 Colorimetric Assay Kit (Biovision, Milpitas, CA, USA) according to the manufacturer’s instructions. Myeloperoxidase (MPO) activity was measured in lung lysates and BAL cell pellets using the EnzChek Myeloperoxidase Activity Assay Kit (Life Technologies, Carlsbad, CA, USA). Total RNA was isolated from lung homogenates and cultured macrophages. Total RNA was isolated (Qiagen, Valencia, CA, USA), and specific transcripts were quantified by real-time PCR using TaqMan FAM-labeled probes (Applied Biosystems, Foster City, CA, USA) as described by Rohani et al.27 (link) Total IL-1β levels were measured using the Mouse IL-1β ELISA Ready-SET-Go! Kit (eBioscience).
Vandivort T.C., Birkland T.P., Domiciano T.P., Mitra S., Kavanagh T.J, & Parks W.C. (2017). Stromelysin-2 (MMP-10) facilitates clearance and moderates inflammation and cell death following lung exposure to long multiwalled carbon nanotubes. International Journal of Nanomedicine, 12, 1019-1031.
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3

Neutrophil Activation by fMLF and mtDNA

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The isolated mouse bone marrow neutrophils were placed in a 24-well plate at a density of 5 × 106 cells in 1 ml of dHBSS. fMLF (1 μM), mtDNA (5 μg/ml) were added to the cells and incubated for 4 h. MPO released into culture medium was determined using EnzChek Myeloperoxidase Activity Assay Kit (Invitrogen). Release of neutrophil elastase was measured by enzyme-linked immunosorbent assay (ELISA) using Mouse neutrophil elastase ELISA Kit (CUSABIO Life science, China).
Wei X., Shao B., He Z., Ye T., Luo M., Sang Y., Liang X., Wang W., Luo S., Yang S., Zhang S., Gong C., Gou M., Deng H., Zhao Y., Yang H., Deng S., Zhao C., Yang L., Qian Z., Li J., Sun X., Han J., Jiang C., Wu M, & Zhang Z. (2015). Cationic nanocarriers induce cell necrosis through impairment of Na+/K+-ATPase and cause subsequent inflammatory response. Cell Research, 25(2), 237-253.
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4

Neutrophil Activation and Secretion Assay

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Mouse bone marrow neutrophils were seeded in 24-well plate with RPMI complete medium at a concentration of 5 × 106/ml. Then, mitochondria (200 µg/ml), mtDNA (2.5 µg/ml) and fMLF (1 µM) were added to the cells, and incubated at 37°C for 2 h. Activated neutrophils released myeloperoxidase (MPO) and elastase into medium. The medium was collected, centrifuged and the supernatants were kept in -80°C. MPO production and elastase release were determined using EnzChek Myeloperoxidase Activity Assay Kit (Invitrogen) and Mouse neutrophil elastase ELISA Kit (CUSABIO Life science, China), respectively.
Yuan X., Nie W., He Z., Yang J., Shao B., Ma X., Zhang X., Bi Z., Sun L., Liang X., Tie Y., Liu Y., Mo F., Xie D., Wei Y, & Wei X. (2020). Carbon black nanoparticles induce cell necrosis through lysosomal membrane permeabilization and cause subsequent inflammatory response. Theranostics, 10(10), 4589-4605.
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5

Myeloperoxidase Activity Assay in Cells

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The MPO activity of cell lysates was measured using an EnzChek Myeloperoxidase Activity Assay Kit (Invitrogen, E33856) following manufacturer’s instructions. Briefly, cells were resuspended in at 5 x 105 cells / ml in PBS and lysed through freeze thaw cycles and 25 μl added to each well of a 384 well dish (PerkinElmer, 6007270). Chlorination was measured by addition of AFP reagent and fluorescence was measured using a BMG PHERAstar at excitation and emission wavelengths of 485 nm and 520 nm, respectively. Mean fluorescence from cell-free wells was subtracted from experiment wells to control for background fluorescence. Experiments were performed on three independent differentiations and three independent donors using at least technical triplicates.
Harper T.C., Oberlick E.M., Smith T.J., Nunes D.E., Bray M.A., Park S., Driscoll C.D., Mowbray S.F, & Antczak C. (2023). GATA1 deletion in human pluripotent stem cells increases differentiation yield and maturity of neutrophils. iScience, 26(10), 107804.
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6

Quantification of Intestinal Inflammation

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Fixed intestines were flat-mounted, photographed, and the total lesion area was quantitated by a blinded scorer using digital morphometry (Scion Image; Scion Corporation or ImageJ; NIH). Photomicrographs were acquired of fixed jejunal samples that were embedded in paraffin and stained with hematoxylin and eosin. Chlorination and peroxidation activity were determined using EnzChek Myeloperoxidase Activity Assay Kit (Invitrogen, Waltham, MA) according to the manufacturer’s instructions. Intestinal IL-23 normalized to protein and serum HMGB1 concentrations were measured by western blot. Serum TNF was measured by ELISA (R&D Systems, Minneapolis, MN). Concentrations of select other mediators of intestinal inflammation were measured by a quantitative multiplexed electrochemiluminescence assay (Rat Discovery Kit, Meso Scale Discovery, Rockville, MD).
Caravaca A.S., Levine Y.A., Drake A., Eberhardson M, & Olofsson P.S. (2022). Vagus Nerve Stimulation Reduces Indomethacin-Induced Small Bowel Inflammation. Frontiers in Neuroscience, 15, 730407.
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