Recombinant rat PEDF (GenBank accession number: NM_177927): Cusabio Biotech Co, Ltd (Wuhan, Hubei, China); The bacterial lipopolysaccharide (LPS) (L4391, O111:B4) and MPO colorimetric activity assay kits: Sigma (St. Louis, MO, USA). The LDH cytotoxicity assay kit and BCA protein concentration determination kit: Beyotime (Shanghai, China); Tissue and cell total protein extraction kits: Sangon Biotech (Shanghai, China). ELISA kits to measure TNF-α, IL-1β, and IL-6: Shanghai Renjie Biotechnology Co., Ltd. (Shanghai, China) [15 (link)]. ELISA kits to measure PEDF: Shanghai Yan Qi Biological Technology Co Ltd. The TUNEL kit, annexin V‑FITC/PI apoptosis detection kit, and DAPI staining solution: KeyGEN Biotech (Nanjing, Jiangsu, China). Anti-rabbit cleaved-caspase-3 (Catalog No. #9661), anti-rabbit p38 MAPK (Catalog No. #8690), anti-rabbit phospho-p38 MAPK (Catalog No. #4511), anti-rabbit NF-κB p65 (Catalog No. #8242), and anti-rabbit phospho-NF-κB p65 (Catalog No. #3033) antibodies: Cell Signaling Technology (Danvers, MA, USA). Anti-rabbit NLRP3 (Catalog No. ab263899), anti-rabbit PPAR-γ (Catalog No. ab178860) antibodies and PPAR-γ inhibitor (GW9662, Catalog No. ab141125): Abcam (Cambridge, MA, USA) [16 (link)]. Anti-rabbit RIP3 (Catalog No. 17563–1-AP) and anti-mouse β-tubulin (Catalog No. 66240–1-Ig) antibodies: Proteintech (Wuhan, Hubei, China).
Mpo colorimetric activity assay kit
Manufactured by Merck Group
Sourced in United States
The MPO colorimetric activity assay kit is a laboratory tool designed to quantify the activity of the enzyme myeloperoxidase (MPO) in biological samples. The kit utilizes a colorimetric detection method to measure MPO activity, providing researchers with a standardized and reliable way to analyze this important enzyme.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Recombinant rat pedf, by Cusabio (1 mentions)
Bca protein concentration determination kit, by Beyotime (1 mentions)
Dapi staining solution, by Keygen Biotech (1 mentions)
Ldh cytotoxicity assay kit, by Beyotime (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Keygen Biotech (1 mentions)
Tunel kit, by Keygen Biotech (1 mentions)
Tnf α, by Abcam (1 mentions)
Tunel detection kit, by Roche (1 mentions)
Ifn γ, by Abcam (1 mentions)
Clariostar plate reader, by BMG Labtech (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Sod activity colorimetric assay kit, by Abcam (1 mentions)
Quantikine immunoassay kit, by R&D Systems (1 mentions)
Gentlemacs octo dissociator with heater, by Miltenyi Biotec (1 mentions)
Milliplex map kit, by Merck Group (1 mentions)
Spectramax plus 384 microplate reader, by Molecular Devices (1 mentions)
14 protocols using mpo colorimetric activity assay kit
1
Investigating PEDF and Inflammatory Pathways
2
Colorimetric Assay of Colonic MPO Activity
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MPO activity in mouse colon tissues was determined using MPO Colorimetric Activity Assay Kits (Sigma-Aldrich) according to the manufacturer's instructions.
3
Quantification of Myeloperoxidase Activity
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Enzymatic activity of the activated PMN’s marker, known as myeloperoxidase (MPO), was quantified in PMNs from WT (n = 6) and FOXO3 KO (n = 6) mice (n = 3 wells per mouse) using MPO Colorimetric Activity Assay Kit according to the manufacturer’s protocol (Sigma, St. Louis, MO, USA). Briefly, collected PMN samples were rapidly homogenized in MPO assay buffer and centrifuged at 13,000× g for 10 min at 4 °C to remove insoluble material. These samples were then plated in a 96-well plate, further assessed, and used for colorimetric detection of MPO activity at 412 nm.
4
Adenosine Receptor Modulation in Tissue
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The A2BR-selective agonist BAY60-6583 and the A2BR-specific antagonist PSB1115 were purchased from Tocris, Bayer Healthcare. The TUNEL detection kit was purchased from Roche Diagnostics GmbH (Penzberg, Germany) and a myeloperoxidase (MPO) colorimetric activity assay kit was purchased from Sigma (MAK 068, USA). The primary antibodies including anti-caspase-3 (ab2302), anti-Ki67 (ab15580), and ELISA measurement kits including IL-6, IL-10, IFN-γ, and TNF-α were purchased from Abcam (Shanghai, China). The BAY60-6583 was dissolved in 100% dimethyl sulfoxide (DMSO) before being diluted in 0.9% saline. An adenosine assay kit (MET-5090) was purchased from Cell Biolabs, Inc. (San Diego, CA, USA).
5
Quantifying Myeloperoxidase Activity
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Myeloperoxidase activity was quantified using an MPO colorimetric activity assay kit (#MAK068; Sigma-Aldrich, St. Louis, MO, USA), according to the manufacture’s instruction. Briefly, a portion of the cecum (50 mg) was homogenized in 4 volumes of MPO Assay Buffer, followed by centrifugation at 13,000× g for 10 min at 4 °C to remove insoluble material. Five µL of supernatant for each sample was added into a well of a 96 wells plate, and its volume was adjusted to a final volume of 50 µL with MPO Assay Buffer. The reaction was stopped after 60 min at room temperature away from light, and the absorbance at 412 nm was measured by a Clariostar Plate reader (BMG LABTECH, Cary, NC, USA). One unit of MPO activity was defined as the amount of enzyme that hydrolyzes the substrate and generates taurine chloramine to consume 1.0 µmole of trinitrobenzene sulphonic acid (TNB) per minute at 25 °C. The results were expressed as milliunit/mL.
6
Colorimetric Assay of Intestinal MPO Activity
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The tissue concentrations of MPO were measured to assess the degree of intestinal inflammation using the MPO colorimetric activity assay kit (#MAK068; Sigma-Aldrich, St. Louis, USA). Fifty microliters of 1:40-diluted colon homogeneity samples was added to 50 microliters of MPO substrate according to the manufacturer’s instructions. The reaction was stopped after 60 min at room temperature away from light, and the absorbance at 412 nm was measured by the microplate reader (Molecular Device, USA). The amount of TNB consumed by the enzyme assay were determined by the difference value between each sample blank and its corresponding samples (ΔA412 = (A412)sample blank-(A412)sample, then we should compare theΔA412 of each sample to the standard curve to get the amount of TNB consumed. The MPO activity was calculated by the following equation: where B = amount of chromophore TNB consumed, and V = sample volume added to well. All the samples were analysed in 3 wells in each experiment, and each experiment was repeated 3 times.
7
Antioxidant and Inflammatory Assays
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Superoxide dismutase (SOD) activity was expressed as the percentage of superoxide inhibition by using a SOD activity colorimetric assay kit (BioVision, USA) in accordance with the manufacturer’s instructions. The respiratory burst (RB) generated by phagocytes was measured via a nitro blue tetrazolium assay (Sigma-Aldrich, USA) as previously described22 . Myeloperoxidase (MPO) activity was analyzed with an MPO colorimetric activity assay kit (Sigma-Aldrich, USA). Antiprotease activity was expressed as the percentage of trypsin inhibition by using the method of Ellis23 . Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total glucose, total protein, triglyceride, and total cholesterols levels were measured with a BS-390 chemistry analyzer (Mindray Bio-Medical Electronics, China) at the Core-Facility Center for Tissue Regeneration, Dong-eui University (Busan, South Korea).
8
Colon Neutrophil Infiltration Quantification
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Infiltration of neutrophils into the colon tissue was measured with the MPO Colorimetric Activity Assay Kit (Sigma). Briefly, the colon was homogenized in MPO assay buffer and supernatants were collected by centrifugation at 13,000× g for 10 min. The supernatant was mixed with MPO assay buffer and MPO substrate, incubated at 25 °C for 120 min, then tetramethylbenzidine (TNB) probe was added. Absorbance was measured at 412 nm using a spectrophotometer (Biotek).
9
Colorimetric Assay for Colonic MPO
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MPO activity was measured using an MPO colorimetric activity assay kit (Sigma–Aldrich). Briefly, the colonic tissues (50 mg) were homogenized in 1 mL of PBS and centrifuged at 13,000×g for 10 min at 4 °C. The supernatant was collected, and the MPO activity was determined according to the manufacturer's instructions. MPO activity is expressed in units per gram of colonic tissue. One unit of MPO activity is defined as the amount of enzyme degrading 1 μmol of peroxide per minute at 25 °C. The concentrations of pro-inflammatory cytokines (IL-6 and TNF-α) in the colonic tissues were also measured by ELISA. The colonic tissues (50 mg) were homogenized in 1 mL of RIPA buffer containing protease inhibitors and centrifuged at 13,000×g for 10 min at 4 °C. The supernatant was collected, and the concentrations of IL-6 and TNF-α were measured using ELISA kits (Invitrogen, Waltham, MA, USA). The total protein concentration was determined by using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).
10
Inflammatory and Apoptotic Markers in Rat Colon
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As instructed, IL-10 was determined in colon tissue homogenates using the Quantikine Immunoassay kit purchased from R&D Systems (Cat. No. R1000). IL-12 p70 was quantified in colon tissue homogenates using a kit purchased from CUSABIO (Wuhan, China; Cat. No. CSB-E07364r). The results are expressed as IL-10/IL-12 p70 ratio. An MPO colorimetric activity assay kit purchased from Sigma-Aldrich was used to determine the activity of myeloperoxidase in the rat colon (Cat. No. MAK068). In this assay, MPO catalyzes the formation of hypochlorous acid, which reacts with taurine to form taurine chloramine. Taurine chloramine reacts with the chromophore TNB, resulting in the formation of the colorless product DTNB. One unit of MPO activity is defined as the amount of enzyme that hydrolyzes the substrate and generates taurine chloramine to consume 1.0 μmole of TNB per minute at 25°C. A colorimetric assay of caspase-1 was performed using a kit supplied by BioVision (Milpitas, CA, USA; Cat. No. K111-25). Caspase-1 activity was spectrophotometrically determined on the basis of the detection of the cleaved p-nitroanilide (p-NA) chromophore at 405 nm.
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