COX-2 inhibition was measured by using a colorimetric COX-2 inhibitor screening assay kit (COX Activity Assay Kit, item No.760151) (Cayman Chemical, USA). The effect of PMG on COX-2 inhibition activity was performed according to the manufacturer's protocol. Cox-2 working solution was prepared by dissolving COX-2 agent in 100 mM Tris-HCl buffer, pH 8.0 at a ratio of 1 : 100. In brief, the reaction mixture containing 150 μl of assay buffer, 10 μl of PMG, 10 μl of heme (Cayman Chemical, USA), 10 μl of COX-II working solution, 20 μl of 10 μM TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride) (Sigma, USA), and 20 μl of 100 μM arachidonic acid (Cayman Chemical, USA) was added to a 96-well microplates and incubated at 25°C for 30 minutes. At the end of the incubation time period, an absorbance at 590 nm was recorded and results were expressed as EC50 [22 (link)]. Indomethacin was used as a positive control.
Tmpd n n n n tetramethyl p phenylenediamine dihydrochloride
Manufactured by Merck Group
Sourced in United States
TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride) is a chemical compound used as a reagent in various laboratory applications. It is a colorless to pale yellow crystalline solid. TMPD is commonly used as an electron transfer agent and a redox indicator in biochemical and analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Arachidonic acid, by Cayman Chemical (2 mentions)
Cox activity assay kit, by Cayman Chemical (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Hematin, by Merck Group (1 mentions)
Amplex red, by Thermo Fisher Scientific (1 mentions)
Adenosine diphosphate (adp), by Merck Group (1 mentions)
Horseradish peroxidase hrp, by Thermo Fisher Scientific (1 mentions)
Malate, by Merck Group (1 mentions)
Succinate, by Merck Group (1 mentions)
Ascorbate, by Merck Group (1 mentions)
Glutamate, by Merck Group (1 mentions)
Amytal, by Merck Group (1 mentions)
Antimycine, by Merck Group (1 mentions)
4 protocols using tmpd n n n n tetramethyl p phenylenediamine dihydrochloride
1
Colorimetric Assay for COX-2 Inhibition
2
Colorimetric COX-II Inhibition Assay
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To assess the cyclo-oxygenase-II (COX-II) inhibition, a colorimetric COX-II inhibitor screening assay kit (Cayman Chemical, USA) was employed. The extract’s anti-inflammatory effect on COX-II inhibition activity was determined following the manufacturer’s protocol. A COX-II working solution was prepared by dissolving the COX-II substance in 100 mM Tris-HCl buffer with a pH of 8.0, at a ratio of 1:100. In summary, a mixture containing 150 µL of assay buffer, 10 µL of the extract, 10 µL of heme (Cayman Chemical, USA), 10 µL of COX-II working solution, 20 µL of 10 µM TMPD (N, N,N’,N’-Tetramethyl-p-phenylenediamine dihydrochloride) (Sigma, USA), and 20 µL of 100 µM arachidonic acid (Cayman Chemical, USA) was added to 96-well microliter plates. The plates were then incubated at 25 °C for 30 min. Subsequently, the absorbance at 590 nm was measured, with indomethacin used as the reference standard [29 (link)]. The results were expressed as EC50 value.
3
Fatty Acid Metabolism Pathway Assay
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Sigma-Aldrich was chosen as the local supplier for the chemicals, drugs, and solvents used in this research. Arachidonic acid (CAT No: 152386) and linoleic acid (CAS No: 60-32-2) are fatty acids that are precursors to lipoxygenase (5-LOX) and cyclooxygenase (COX-2), respectively. Glutathione (GSH) (CAS 72-16-6), N, N,N,N-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) (CAS 637-01-4), and hematin (CAS No. 15485-92-6) from Sigma-Aldrich serve as cofactors and indicators. Analytical grade solvents were used.
4
Mitochondrial Respiratory Function in Rats
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Synthetic THC (25 mg/mL in ethanol), mitochondrial complexes substrates and/or inhibitors such as glutamate, malate, amytal, ADP, succinate, ascorbate, antimycine, and N,N,N′,N′-tetramethyl-p-phenylenediaminedihydrochloride (tmpd) were acquired from Sigma-Aldrich, France. THC was successively diluted in ethanol as needed and Amplex Red and horseradish peroxidase (HRP) were acquired by Invitrogen. All other chemicals used were of the highest grade commercially available.
Ten male Wistar rats (weight 438 to 500 grams; age 13 weeks) were housed in a neutral temperature environment (22° ± 2°C), on a 12 : 12 hours photoperiod, and were provided food and water ad libitum. This investigation was carried out in accordance with the Helsinki accords for human treatment of animals during experimentation. Rats were submitted to general anesthesia with 3% isoflurane and oxygen (2 L/min) in an induction chamber (Minerve, Esternay, France) and were then decapitated. Brains were excised and cleaned and then immediately used for the study of respiratory parameters.
Ten male Wistar rats (weight 438 to 500 grams; age 13 weeks) were housed in a neutral temperature environment (22° ± 2°C), on a 12 : 12 hours photoperiod, and were provided food and water ad libitum. This investigation was carried out in accordance with the Helsinki accords for human treatment of animals during experimentation. Rats were submitted to general anesthesia with 3% isoflurane and oxygen (2 L/min) in an induction chamber (Minerve, Esternay, France) and were then decapitated. Brains were excised and cleaned and then immediately used for the study of respiratory parameters.
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