Tissue GSH and GSSG levels were measured in whole brain, the cortex and hippocampus using a microplate-adapted fluorometric o-phthalaldehyde (OPA) method (Senft et al., 2000 (link)). Fluorescence was determined with 365 nm excitation and 430 nm emission filters in a DTX 800/880 Multimode Detector (Beckman Coulter, Fullerton, CA, USA).
Dtx 800 880 multimode detector
Manufactured by Beckman Coulter
Sourced in United States
The DTX 800/880 Multimode Detector is a versatile laboratory instrument capable of performing various detection techniques. It is designed to measure multiple analytical parameters, including absorbance, fluorescence, and luminescence, within a single platform. The device is equipped with a flexible optical system and can accommodate a wide range of sample types and formats. The core function of the DTX 800/880 Multimode Detector is to provide accurate and reliable data acquisition for researchers and analysts working in various scientific disciplines.
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7 protocols using dtx 800 880 multimode detector
1
Quantifying Brain Glutathione Levels
2
Quantifying eGFP-Bait Protein Binding
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To estimate the approximate efficiency of binding of eGFP-tagged bait proteins to the anti-GFP beads, the fluorescence intensity was measured. In brief, cell lysates derived from recombinant M. smegmatis strains expressing eGFP fusion proteins were prepared and pre-cleared by centrifugation and the flow-through after binding to the column was diluted 1∶1 in IPP150 buffer and transferred onto a 96-well black solid plate (Nunc, Thermo Scientific). Cell lysate from the M. smegmatis mc2155 parent was used as a background control. Lysates were prepared from approximately the same number of cells as measured by cell pellet weight. Fluorescence counts were measured using Beckman Coulter DTX 800/880 Multimode Detector and Multimode Detection software. Excitation at 485 nm and emission at 535 nm was used, with a data integration time of 1 s. The relative binding efficiency was calculated by dividing the fluorescence intensity of flow-through by the intensity of the lysate before binding to the column, multiplied by 100%.
3
Fluorometric Glutathione Assay
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Glutathione and GSH disulfide (GSSG) contents were assayed using the fluorometric o-phthalaldehyde (OPA) method (Senft et al., 2000 (link)), which was previously proven in our laboratory by HPLC [18], using 96-well black microplates. Fluorescence readings were taken at 365-nm excitation and 430-nm emission with a Beckman Coulter DTX 800/880 Multimode Detector (Beckman Coulter, Fullerton, CA, United States). The final values were calculated as the fluorescence of unit B minus the fluorescence of unit A (UFB-UFA = UFF).
4
Quantifying Ferroptosis in Liver
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The levels of free Fe 2+ were measured as a marker of ferroptosis in liver homogenates using an Iron Assay Kit (Colorimetric) (Abcam Cat No. ab83366). The absorbance of the complex formed by Fe 2+ with the iron probe was read at 593 nm in a DTX 800/880 Multimode Detector (Beckman Coulter, Fullerton, CA, USA).
5
Microplate-based Glutathione Quantification
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GSH levels were measured using a microplate-adapted fluorometric o-phthalaldehyde (OPA) method [26] (link). Fresh tissue samples were homogenized in 10 volumes of buffer A (154 mM KCl, 5 mM DTPA, and 0.1 M K 2 PO 4 ), then, an equal volume of buffer B (40 mM HCl, 10 mM DTPA, 20 mM ascorbic acid and 10% TCA) was added. The samples were centrifuged at 14,000 × g for 30 min, and supernatants were filtered using Millipore PTFE 0.45 μm filters. GSH levels were determined by fluorescence with 365 nm/430 nm (excitation/emission) filters in a DTX 800/880 Multimode Detector (Beckman Coulter, Fullerton, CA, USA).
6
Fluorometric Measurement of Reduced Glutathione
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Reduced GSH levels were measured in fresh tissue preparations of the cortex and hippocampus using a microplate-adapted fluorometric o-phthalaldehyde (OPA) method (Silva-Adaya et al. 2020 (link)). The method is based on the conjugation of reduced GSH with o-phthalaldehyde (OPA), forming a stable and fluorescent isoindole derivate (Senft et al. 2000 (link)). GSH levels were determined by fluorescence with 365 nm/430 nm (excitation/emission) filters in a DTX 800/880 Multimode Detector (Beckman Coulter, Fullerton, CA, USA).
7
Cortical GSH Quantification via OPA Assay
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The levels of reduced GSH were measured in the cortex, hippocampus, striatum, and cerebellum using a microplate-adapted fluorometric o-phthalaldehyde (OPA) method (Ramos-Chávez et al., 2015 (link)). The method is based on the GSH reaction with o-phthaldialdehyde (OPA) to form a highly stable and fluorescent isoindole derivative. Briefly, wet tissue was homogenized in 10 volumes of ice-cold buffer (154 mM KCl, 5 mM diethylenetriaminepentaacetic acid, and 0.1 M potassium phosphate buffer, pH 6.8). Immediately thereafter, equal volumes of cold acid buffer [40 mM HCl, 10 mM DTPA, 20 mM ascorbic acid, and 10% trichloroacetic acid (TCA)] were added to one volume of homogenate. Two microliters of supernatant was used for GSH determination. Fluorescence was determined with 365 nm excitation and 430 nm emission filters in a DTX 800/880 Multimode Detector (Beckman Coulter, Fullerton, CA, USA).
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