Brain tissue homogenates in 25 mM HEPES buffer (pH 7.4) containing 250 mM sucrose were centrifuged at 1000× g for 5 min at 4 °C. The supernatants were collected to measure the glutathione concentration using the Fluorimetric Glutathione Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions.
Fluorimetric glutathione assay kit
Manufactured by Merck Group
Sourced in United States
The Fluorimetric Glutathione Assay Kit is a laboratory equipment product designed to quantify the levels of glutathione, a key antioxidant, in biological samples. The kit utilizes a fluorometric detection method to accurately measure glutathione concentrations.
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4 protocols using fluorimetric glutathione assay kit
1
Glutathione Quantification in Brain Tissue
2
Fluorimetric Glutathione Assay Protocol
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Cells were washed in PBS, detached by trypsin and washed with PBS. Cells were then lysed and assayed in accordance with the protocol for Fluorimetric Glutathione Assay kit (CS1020, Sigma). GSH levels in the cells were determined by use of a GSH standard curve, and all samples were measured in triplicate. The readout was performed using Tecan Spark 10 microplate reader, and the data was analysed using GraphPad Prism 7.
3
Quantitative Assays for NADPH and GSH
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NADPH levels were determined using a colorimetric NADP+/NADPH Quantitation Kit (BioVision) according to the manufacturer's protocol. The signal at 450 nm was recorded using a Victor 3 microplate reader and normalized to protein concentration. GSH levels were measured using a Glutathione Assay Fluorimetric Kit (Sigma-Aldrich) according to the manufacturer's procedure. Fluorimetric signal was recorded on a Victor 3 microplate reader and normalized to protein concentration.
4
Quantifying NADPH and Glutathione
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The colorimetric NADP+/NADPH Quantitation Kit (BioVision) was applied to determine NADPH levels according to the manufacturer’s protocol. The 450 nm signal was recorded using the Victor 3 microplate reader and then normalized to protein concentration. The Glutathione Assay Fluorimetric Kit (Sigma-Aldrich) was used to measure GSH levels according to the manufacturer’s procedure. The signal was recorded on the Victor 3 microplate reader and then normalized to protein concentration.
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