Glutathione measurements were performed in total homogenate of mouse tissues. Tissues were homogenized in sodium phosphate buffer (A) (100 mm sodium phosphate, 5 mm EDTA-Na2, pH 8.0) and deproteinized using ice-cold TCA 40% and centrifuged at 20 000g for 15 min. For GSH measurement, the supernatant was incubated with a solution of (A) and orthophthalaldehyde/ethanol (B) (1 mg/ml) for 15 min at room temperature. The fluorescence of the samples was measured at 340 nm excitation and 420 nm emission wavelengths in a spectrofluorometer plate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). For GSSG measurement, the supernatant was preincubated with N-ethylmaleimide solution (5 mg/ml) for 40 min then alkalinized with 0.1 N NaOH (C). Aliquots of these mixtures were incubated with (B) and (C) for 15 min at room temperature. Finally, the fluorescence was measured as before. GSH and GSSG concentrations were calculated according to standard curves that we previously prepared, and the levels of GSH and GSSG were expressed in nmol/mg protein (8 (link)).
Spectrofluorometer plate reader
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The Spectrofluorometer plate reader is a laboratory instrument used to measure the fluorescence of samples in a microplate format. It is designed to quantify the fluorescence intensity of multiple samples simultaneously.
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5 protocols using spectrofluorometer plate reader
1
Glutathione Measurement in Mouse Tissues
2
Glutathione Measurement in Tissues and Cells
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Glutathione measurements were performed in cytosol and mitochondrial fractions of mouse tissues, as well as in Hepa1c1c7 cells and human skin fibroblasts, which were cultured in Opti‐MEM medium lacking FBS for 72 h in order to avoid influence of the GSH contained in the FBS.
Glutathione was measured by an established fluorometric method (Hissin & Hilf,1976 ). Mitochondrial pellets were resuspended in sodium phosphate buffer (A) (100 mM sodium phosphate, 5 mM EDTA‐Na2, pH 8.0). Mitochondrial and cytosolic fractions were deproteinized with ice‐cold TCA 40% and centrifuged at 20,000 g for 15 min. For GSH measurement, the supernatant was incubated with a solution of (A) and orthophthalaldehyde/ethanol (B) (1 mg/ml) for 15 min at room temperature. The fluorescence of the samples was then measured at 340 nm excitation and 420 nm emission wavelengths in a spectrofluorometer plate reader (Bio‐Tek Instruments Inc., Winooski, VT, USA). For GSSG measurement, the supernatant was preincubated with N‐ethylmaleimide solution (5 mg/ml) for 40 min and then alkalinized with 0.1 N NaOH (C). Aliquots of these mixtures were then incubated with (B) and (C) for 15 min at room temperature. The fluorescence was then measured as before. GSH and GSSG concentrations were calculated according to standard curves prepared, and the levels of GSH and GSSG are expressed in nmol/mg protein.
Glutathione was measured by an established fluorometric method (Hissin & Hilf,
3
LDH Activity Monitoring in OA Chondrocytes
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The lactate dehydrogenase (LDH) detection kit (Roche Diagnostics, Indianapolis, IN, USA) was used to monitor the activity of LDH in the OA-derived chondrocytes after 2, 12 and 48 h. One hundred microliters of the medium was discarded from each well, and then 50 μl of 2% Triton X-100 solution was added to lyse the cells. The samples were incubated in the dark for 30 min at room temperature, and then were detected by fluorescence (490 nm) using a BioTek spectrofluorometer plate reader with KC4 analysis software (BioTek, Winooski, VT, USA).
4
Glutathione Measurement in Erythrocytes
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For glutathione measurement, 10 μL of the supernatant of hemolyzed erythrocytes were incubated with 10 μL of ethanol ophthalaldehyde solution (1 g/L) and 180 μL phosphate buffer A (100 mM sodium phosphate, 5 mM EDTA-Na2, pH 8.0) for 20 min at room temperature. Then, the fluorescence of the samples was measured at 350 nm excitation and 420 nm emissions in a plate-reader spectrofluorometer (Bio-Tek Instruments, Inc., Winooski, VT, USA) [72 (link)]. For glutathione disulfide measurement, 200 μL aliquots of supernatants were preincubated with 80 μL N-ethylmaleimide solution (5 g/L in distilled water) for 40 min at room temperature and then alkalinized with 720 μL sodium hydroxide 0.1 N. Aliquots of 30 μL were then incubated with 10 μL ophthalaldehyde solution and 160 μL sodium hydroxide 0.1 N for 25 min at room temperature, and the fluorescence was then measured [25 (link)]. Glutathione concentrations were calculated according to standard curves previously prepared. Levels are expressed in μmoL/g Hb.
5
Fluorometric Assay for Glutathione Redox Status
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Glutathione (GSH) and glutathione disulfide (GSSG) were measured using an established fluorometric method [31 ]. Small intestine mitochondria were sonicated in ice-cold 50 mM Tris-HCl buffer (pH 7.4), and then centrifuged at 800 × g for 10 min at 4°C. Aliquots of the supernatant were deproteinized using ice-cold 10% trichloroacetic acid, and then centrifuged at 20,000 × g for 15 min. For GSH measurement, supernatant aliquots were incubated for 15 minutes at room temperature with an ortho-phthalaldehyde/ethanol solution (1 mg/mL) and phosphate buffer (100 mM sodium phosphate and 5 mM EDTA-Na2, pH 8.0). Then the sample fluorescence was measured at 340 nm excitation and 420 nm emission wavelengths using a plate-reader spectrofluorometer (Bio-Tek Instruments, Inc., Winooski, VT, USA).
For GSSG measurement, supernatant aliquots were preincubated with N-ethylmaleimide solution (5 mg/mL) at room temperature for 40 min, and then alkalinized with 0.1 N NaOH. Aliquots of this mixture were then incubated for 15 minutes at room temperature with ortho-phthalaldehyde solution (1 mg/mL) and 0.1 N NaOH, and the sample fluorescence was then measured. GSH and GSSG concentrations were expressed as nmol/mg protein.
For GSSG measurement, supernatant aliquots were preincubated with N-ethylmaleimide solution (5 mg/mL) at room temperature for 40 min, and then alkalinized with 0.1 N NaOH. Aliquots of this mixture were then incubated for 15 minutes at room temperature with ortho-phthalaldehyde solution (1 mg/mL) and 0.1 N NaOH, and the sample fluorescence was then measured. GSH and GSSG concentrations were expressed as nmol/mg protein.
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