On day 10, 5 plates of worms were washed with M9 buffer and incubated with 100 μM Amplex Red probe (Invitrogen, CA, USA) in Krebs-Ringer Phosphate buffer (145 mM NaCl, 5.7 mM Na2PO4, 4.86 mM KCl, 0.54 mM CaCl2, 1.22 mM MgSO4 and 5.5 mM glucose, pH 7.4) containing 0.2 U ml−1 of horseradish peroxidase. After occasional mixing in the dark for 3 h, fluorescence intensity was determined using a fluorescence plate reader (Synergy H1, BioTek, VT, USA; excitation: 571, emission: 585) by normalization using protein input with BCA protein assay (Pierce, IL, USA). Three biological replicates of the measurement were performed.
Amplex red probe
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Amplex Red probe is a fluorogenic indicator used for the detection and quantification of hydrogen peroxide (H2O2) in biological samples. It functions by reacting with H2O2 in the presence of the enzyme horseradish peroxidase to produce the fluorescent product resorufin, which can be measured using a fluorometer.
Automatically generated - may contain errors
Lab products found in correlation
Synergy h1, by Agilent Technologies (1 mentions)
Varioskan flash, by Thermo Fisher Scientific (1 mentions)
Fluoroskan ascent fl, by Thermo Fisher Scientific (1 mentions)
Hysox, by Goryo Chemical (1 mentions)
Oxiorange, by Goryo Chemical (1 mentions)
Axio observer, by Zeiss (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Sodium citrate, by Merck Group (1 mentions)
Heparin, by Merck Group (1 mentions)
6 protocols using amplex red probe
1
Quantifying Worm Peroxidase Activity
2
Quantifying Cellular Hydrogen Peroxide
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H2O2 assessment was performed in intact, seeded cells. 1.25 × 105 cells were incubated in 100 μM AmplexRed probe (Invitrogen) in Krebs-Ringer phosphate buffer (145 mM NaCl, 5.7 mM Na2PO4, 4.86 mM KCl, 0.54 mM CaCl2, 1.22 mM MgSO4 and 5.5 mM glucose), containing horseradish peroxidase (0.5 U/ml). Luminescence was recorded for 2 h at 30 min intervals using a Varioskan Flash (Thermo Scientific, Rockford, IL, USA) fluorimeter (excitation: 538 nm, emission: 604 nm), and the slopes were compared with those obtained with H2O2 (0–10 µM) for the calculations.
3
ROS and Glutathione Measurement in Cortical Neurons
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For ROS assessment in cortical neurons, whole cell-derived H2O2 was measured, in intact cells, using the fluorescent AmplexRed probe (Invitrogen), 1.5 × 104 cells were incubated in 100 μM AmplexRed in Krebs–Ringer Phosphate buffer (145 mM NaCl, 5.7 mM Na2PO4, 4.86 mM KCl, 0.54 mM CaCl2, 1.22 mM MgSO4, and 5.5 mM glucose, pH 7.4), and the luminescence recorded for 45 min at 1-min intervals using a Fluoroskan Ascent FL (Thermo Scientific, Rockford, IL, USA) fluorimeter (excitation: 538 nm, emission: 604 nm), and the slopes were used for calculations.
Glutathione levels were also determined, in cortical neurons, by adding 1% (wt/vol) of sulfosalicylic acid and an equal volume of NaOH 0.1 M to the cells. Cell lysates were centrifuged at 13,000×g for 5 min at 4 °C, and the supernatants were used for the determination of total glutathione (GSH + 2xGSSG), as described previously (Griffith 1980 (link)).
Glutathione levels were also determined, in cortical neurons, by adding 1% (wt/vol) of sulfosalicylic acid and an equal volume of NaOH 0.1 M to the cells. Cell lysates were centrifuged at 13,000×g for 5 min at 4 °C, and the supernatants were used for the determination of total glutathione (GSH + 2xGSSG), as described previously (Griffith 1980 (link)).
4
Quantifying Intracellular Reactive Oxygen Species
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The concentration of H2O2 in pure water and culture medium generated by EES treatment was measured using the Amplex Red probe (Invitrogen, Carlsbad, CA, USA). EES treatment was performed on 1.5 ml pure water or culture medium without cells, and 50 μl of the treated solutions was transferred to individual wells of a 96-well transparent plate. To generate a standard curve, H2O2 solution of 0 to 10 μM was prepared. After adding the Amplex Red reagent to each well, the microplate was incubated at room temperature for 30 min. Then, fluorescence was measured using a multiwell plate reader (TECAN Infinite 200 PRO, Männedorf, Switzerland) with excitation/emission filters of 550/585 nm.
Detection of other intracellularly induced ROS was also performed using fluorescent probes. Cells were cultured for 3 days and then stained with OxiOrange (excitation: 543 nm, emission: 577 nm; Goryo Chemical, Sapporo, Japan) or HySOx (excitation: 555 nm, emission: 575 nm; Goryo Chemical), which react with intracellular HO· and hypochlorous acid (HClO), respectively, and emit strong fluorescence. After staining for 30 min at 37°C, cells were washed with PBS, and EES treatment was conducted with 0.5 ml culture medium, and then intracellularly induced ROS were observed by fluorescence microscopy (Axio Observer, Carl Zeiss, Jena, Germany). Nuclei were stained with DAPI (4′6-diamidino-2-phenylindole).
Detection of other intracellularly induced ROS was also performed using fluorescent probes. Cells were cultured for 3 days and then stained with OxiOrange (excitation: 543 nm, emission: 577 nm; Goryo Chemical, Sapporo, Japan) or HySOx (excitation: 555 nm, emission: 575 nm; Goryo Chemical), which react with intracellular HO· and hypochlorous acid (HClO), respectively, and emit strong fluorescence. After staining for 30 min at 37°C, cells were washed with PBS, and EES treatment was conducted with 0.5 ml culture medium, and then intracellularly induced ROS were observed by fluorescence microscopy (Axio Observer, Carl Zeiss, Jena, Germany). Nuclei were stained with DAPI (4′6-diamidino-2-phenylindole).
5
Pancreatic Islets Hydrogen Peroxide Assay
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Groups of 120 pancreatic islets were placed in a microtube and pre-incubated for 30 min at 37 °C with in Krebs-Henseleit buffer with 5.6 mM glucose and 0.1% albumin. After this period, the islets were incubated for 1 hour at 37 °C with 2.8 or 16.7 mM glucose. Then, the samples were sonicated and incubated for 30 minutes with 50 μM Amplex Red probe (Life Technologies, Eugene, OR, USA), a specific marker for hydrogen peroxide (H2O2). The samples were placed into a 96-well plate for absorbance reading at 560 nm in a microplate reader (Biotek, Winooski, VT, USA).
6
HDL Cholesterol Determination via Heparin-Citrate Precipitation
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Heparin, purchased from Panpharma (Luitre-Fougeres, France) (25,000 IU/5 ml) and sodium citrate from Merck Millipore (Billerica, MA, USA) were used to prepare an LDL precipitation solution of 100 IU Heparin/ml in citrate buffer 0.064 M. The HDL assay solution was obtained from Merck Millipore (1.4 mmol/l phosphotungstic acid, 8.6 mmol/l magnesium chloride). The Amplex Red probe from Life Technologies (Grand Island, NY, USA) was used to prepare a stock solution of 20 mM in DMSO, from which a working solution of 300 µM was prepared in 1X phosphate-buffered saline (PBS) (Biochrom AG, Berlin, Germany).
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