For quantification of H2O2, 50 mg of milkfish livers were homogenized in PBS solution and then centrifuged for 15 min at 1000 × g. The H2O2 contents were subsequently determined using the hydrogen peroxide colorimetric/fluorometric assay kit (Biovision, Bilpitas, CA, USA). We added 10 μL samples into each well of the 96-well plate, and brought the volume to 50 μL with the H2O2 assay buffer. Different concentrations of H2O2 (0, 1, 2, 3, 4, 5 nmol well−1) were used as the standard for calculation. The VERSAmax microplate reader (Molecular Devices, Sunnyvale, CA, USA) with the wavelength of OD570nm was used to measure the H2O2 content.
Hydrogen peroxide colorimetric fluorometric assay kit
Manufactured by Abcam
Sourced in United States
The Hydrogen peroxide colorimetric/fluorometric assay kit is a laboratory equipment designed to quantitatively detect and measure the levels of hydrogen peroxide in samples. The kit utilizes a colorimetric or fluorometric detection method to provide a simple and reliable analysis of hydrogen peroxide.
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Lab products found in correlation
Ms 222, by Merck Group (3 mentions)
Collagenase, by Fujifilm (2 mentions)
Biotinylated anti rabbit igg, by Vector Laboratories (2 mentions)
Streptavidin biotin complex peroxidase kit, by Nacalai Tesque (2 mentions)
Protein assay cbb solution, by Nacalai Tesque (2 mentions)
Atp bioluminescence assay kit cls 2, by Merck Group (2 mentions)
Water soluble progesterone, by Merck Group (2 mentions)
Hydrogen peroxide, by Nacalai Tesque (2 mentions)
Cover slips for microscopy, by Matsunami (2 mentions)
Slide glasses, by Matsunami (2 mentions)
Versamax microplate reader, by Molecular Devices (1 mentions)
Anti vimentin, by Abcam (1 mentions)
5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (1 mentions)
Lucigenin, by Merck Group (1 mentions)
Peroxidase affinipure goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Anti mmp9, by Proteintech (1 mentions)
Anti bcl 2, by Proteintech (1 mentions)
Anti mmp 3, by Abcam (1 mentions)
Anti p38, by Abcam (1 mentions)
Anti jnk, by Abcam (1 mentions)
12 protocols using hydrogen peroxide colorimetric fluorometric assay kit
1
Quantification of H2O2 in Milkfish Liver
2
Assaying PO Activity and ROS Levels in Insect Immune Response
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PO activity in insects was assayed as described previously [57 (link)]. Samples included a time course of infection, i.e., 3, 6, 9, 12, and 18 h after intra-hemocoel injection of 2 μL conidial suspension (107 conidia / mL), as well as at 12 h after intra-hemocoel of (i) UV-killed wild-type conidia (UKC, 5 × 106 spores / mL), (ii) purified BbEng1 protein (0.5 μM) + UKC, (iii) heat-inactivated BbEng1 protein (iBbEng1, 0.5 μM) + UKC. Larvae injected with 2 μL of 0.85%NaCl and UKC + 0.5 μM BSA were used as controls. ROS levels in insect hemolymph were examined by quantification of H2O2 content at 3, 6, 9, 12 and 24 h after intra-hemocoel injection of 2 μL conidial suspension (107 conidia / mL) using the OxiRed Probe (BioVision) and a hydrogen peroxide colorimetric / fluorometric assay kit (BioVision) as described previously [12 (link),58 (link)].
3
Oxidative Stress Biomarker Assay Protocol
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Griess reagent, 5,5’-Dithiobis (2-nitrobenzoic acid) and Lucigenin were purchased from Sigma-Aldrich. Hydrogen peroxide 33% was procured from PanReac AppliChem. The following antibodies were used in this work: Anti-fibronectin was obtained from GeneTex. Anti-MMP-9 and anti-Bcl2 were obtained from Proteintech. Anti-vimentin, anti-MMP-3, Anti-Akt, Anti-p38, Anti-Cleaved PARP1, Anti-p53 (acetyl K382), Anti-JNK, Anti-alpha smooth muscle Actin, Anti-NF-kB p65 (phospho S536), and anti-β catenin were all obtained from Abcam (Cambridge, UK). Anti-cleaved caspase-3, anti-cleaved aspase-9, and anti-cleaved caspase-8 were obtained from Cell Signaling. Rabbit Polyclonal JNK1/2/3 was obtained from ORIGENE. Anti-beta Actin was obtained from Thermo Fisher Scientific. Peroxidase AffiniPure Goat Anti-Rabbit IgG and Peroxidase AffiniPure Goat Anti-Mouse IgG were obtained from Jackson ImmunoResearch Inc. The following ELISA kits were used in this work: nitrotyrosine ELISA kit (Abcam, ab113848), Hydrogen Peroxide Colorimetric/Fluorometric assay kit (Biovision, K265-200), malondialdehyde assay kits (Northwest Life Sciences Specialties, NWK-MDA01) and Superoxide Dismutase assay kit (Cayman chemical, 706002).
4
Assay Reagents for Oxidative Stress
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Water-soluble progesterone (PG), anesthetic MS-222 and apocynin were obtained from Sigma (St. Louis, MO, USA). Collagenase (280 U/mg) and catalase (11,000 U/mg) were purchased from Wako (Osaka, Japan), hCG was from Teikoku Zoki (Tokyo, Japan). Fluorogenic caspase-3 substrate IV was purchased from Calbiochem (La Jolla, CA, USA). Hydrogen peroxide colorimetric/fluorometric assay kit was from BioVision (Milpitas, CA, USA). Polyclonal anti-MAPK and anti-pMAPK antibodies were from Cell Signaling (Beverly, MA, USA), biotinylated anti-rabbit IgG was from Vector Laboratories (Burlingame, CA, USA). The Streptavidin Biotin Complex Peroxidase Kit, protein assay CBB solution, SOD (5000 U/mg) and BHA were from Nacalai Tesque (Kyoto, Japan). 2′,7′-dichlorofluorescein diacetate (DCFDA) Cellular ROS Detection Assay Kit was obtained from Abcam (Cambridge, UK), EUK 134 and MITO-TEMPO were ordered from Santa Cruz (Santa Cruz Biotechnology, Dallas, TX, USA). Other chemicals were obtained from Wako and Nacalai Tesque.
5
Quantifying Cellular Hydrogen Peroxide
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Anesthetic MS-222, water-soluble progesterone, and ATP Bioluminescence Assay Kit CLS II were purchased from Sigma (St. Louis, MO). Collagenase (280 U/mg) was obtained from Wako (Osaka, Japan) and human chorionic gonadotropin was from Teikoku Zoki (Tokyo Japan). Hydrogen peroxide, Sudan Black B (SBB), and protein assay CBB solution were from Nacalai Tesque (Kyoto, Japan). Hydrogen peroxide colorimetric/fluorometric assay kit was from BioVision (Milpitas, CA). Other chemicals were obtained from Wako and Nacalai Tesque. Slide glasses and cover slips for microscopy were purchased from Matsunami Glass (Osaka, Japan).
6
Progesterone-Induced Apoptosis Assay
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Water-soluble progesterone (PG), anesthetic MS-222 and ATP Bioluminescence Assay Kit CLS II were purchased from Sigma (St. Louis, MO, USA). hCG was from Teikoku Zoki (Tokyo, Japan) and collagenase (280 U/mg) was obtained from Wako (Osaka, Japan). The hydrogen peroxide colorimetric/fluorometric assay kit was from BioVision (Milpitas, CA, USA). Fluorogenic caspase-3 substrate IV was purchased from Calbiochem (La Jolla, CA, USA). Polyclonal anti-cyclin B2 antibody was ordered from Santa Cruz (Santa Cruz Biotechnology, Dallas, TX, USA), biotinylated anti-rabbit IgG was from Vector Laboratories (Burlingame, CA, USA). The Streptavidin Biotin Complex Peroxidase Kit, protein assay CBB and hydrogen peroxide were from Nacalai Tesque (Kyoto, Japan). MitoTracker Deep Red FM was from ThermoFisher (Waltham, MA, USA). Luciferase control RNA and luciferase assay system were from Promega (Madison, WI, USA). Other chemicals were obtained from Wako and Nacalai Tesque. Slide glasses and cover slips for microscopy were purchased from Matsunami Glass (Osaka, Japan).
7
Calcium Chelators Modulate Peroxide-Induced Oocyte Changes
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Hydrogen peroxide was added at a final concentration of 10 mM to the oocytes matured in vitro for 10–12 h in the presence of progesterone. The cells were washed with OR-2 buffer before peroxide administration to remove the hormone. The precise concentration of hydrogen peroxide was determined by titration using the hydrogen peroxide colorimetric/fluorometric assay kit from BioVision, according to the manufacturer’s manual. To prepare 50 mM stock solutions of BAPTA (tetrasodium salt) and BAPTA-AM, the drugs were dissolved in water and DMSO, respectively. The chelators were added to eggs at a final concentration of 100 μM 30 min before hydrogen peroxide administration. At the dilution used, DMSO had no effect on egg viability. CaCl2 was excluded from the egg incubation media OR-2 in the experiments with calcium chelators.
8
Quantifying Extracellular H2O2 and Catalase
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To quantify extracellular H2O2 and catalase, T-G1 was cultured in liquid PYG media with 100 μM MnSO4 at pH 5.5 and pH 7.2. Spent media were sampled at exponential and stationary phases, after centrifugation at 1000 ×g for 5 min. The quantity of H2O2 in the supernatant of spent media was measured using the Hydrogen Peroxide Colorimetric/Fluorometric Assay Kit (BioVision, USA) according to the manufacturer’s protocol. The absorbance at 570 nm was measured with a Synergy H4 Hybrid Reader (BioTek). Catalase activity in the supernatant of spent media was measured at exponential phase and stationary phase with 10 μM H2O2 by Amplex® Red Catalase Assay Kit (Life Technology) according to the manufacturer’s protocol and measuring absorbance at 560 nm. All samples were analyzed in triplicate.
9
Quantification of Neuronal H2O2 Production
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To detect H2O2 production, primary cortical neurons were cultured in 24-well plates for 5 d. After POM pretreatment and H2O2-exposure, cells were washed with HBSS and equal volumes of lysates were quantified with red fluorescence (Ex/Em = 535/587 nm) using a Hydrogen Peroxide Colorimetric/Fluorometric Assay kit (BioVision) according to the manufacturer’s instructions.
10
Intracellular Hydrogen Peroxide Quantification
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Intracellular concentration of hydrogen peroxide was determined using the hydrogen peroxide colorimetric/fluorometric assay kit (BioVision), according to the manufacturer’s manual. Caspase activity assay was performed as described previously [30 (link)]. Protein content in the cytosolic fraction of oocytes and eggs was determined with the CBB protein assay.
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