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Amplite® Fluorimetric Glutathione Assay Kit *Green Fluorescence*
The monitoring of reduced and oxidized glutathione (GSH) in biological samples is essential for evaluating the redox and detoxification status of cells and tissues in relation to the protective role of glutathione against oxidative and free-radical-mediated cell injury. Cysteine metabolism disorders include cystinosis, an autosomal recessive disease produced by a defect in lysosomal transport, and cystinuria, a common heritable disorder of amino acid transport. Cysteine is unique among the amino acids found in proteins. There are a few reagents or assay kits available for quantitating thiols in biological systems. However, all the commercial kits either lack sensitivity or have tedious protocols. Our Amplite® Fluorimetric Glutathione Qutitation Kit provides an ultrasensitive fluorimetric assay to quantitate GSH in sample. The kit uses a proprietary non-fluorescent dye that becomes strongly fluorescent upon reacting with thiol. The kit provides a sensitive, one-step fluorimetric method to detect as little as 1 picomole of cysteine or GSH in a 100 µL assay volume. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. Its signal can be easily read using a fluorescence microplate reader.
Example protocol
AT A GLANCE
Protocol Summary
- Prepare GSH working solution (50 µL)
- Add GSH standards or test samples (50 µL)
- Incubate at RT for 10 to 60 minutes
- Monitor the fluorescence increase at Ex/Em = 490/525 nm (Cutoff = 515 nm)
Important Note
Thaw all the kit components at room temperature before starting the experiment.
CELL PREPARATION
For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles
GSH standard solution (1 mM)
Add 200 µL of Assay Buffer (Component B) into the vial of GSH Standard (Component C) to make 1 mM (1 nmol/µL) GSH standard solution.
Thiolite™ Green stock solution (100X)
Add 100 µL of DMSO (Component D) into the vial of Thiolite™ Green (Component A) to make 100X Thiolite™ Green stock solution. Note: Alternatively, if precipitation is observed while making working solution, one can make 50X stock solution using 200 µL DMSO solution.
PREPARATION OF STANDARD SOLUTIONS
For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/10055
https://www.aatbio.com/tools/serial-dilution/10055
GSH standard
Add 30 μL of 1 mM (1 nmol/µL) GSH standard solution to 970 μL of Assay Buffer (Component B) to generate 30 μM (30 pmol/µL) GSH standard solution. Take 30 μM (30 pmol/µL) GSH standard solution and perform 1:3 serial dilutions to get serially diluted GSH standards (SD7-SD1) with Assay Buffer (Component B). Note: Diluted GSH standard solution is unstable. Use within 4 hours.PREPARATION OF WORKING SOLUTION
Add 50 μL of 100X Thiolite™ Green stock solution into 5 mL of Assay Buffer (Component B) and mix well to make GSH working solution.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of GSH standards and test samples in a solid black 96-well microplate. SD = GSH Standards (SD1 - SD7, 0.01 to 10 µM); BL=Blank Control; TS=Test Samples
| BL | BL | TS | TS |
| SD1 | SD1 | ... | ... |
| SD2 | SD2 | ... | ... |
| SD3 | SD3 | ||
| SD4 | SD4 | ||
| SD5 | SD5 | ||
| SD6 | SD6 | ||
| SD7 | SD7 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| SD1-SD7 | 50 µL | Serial Dilutions (0.01 to 10 µM) |
| BL | 50 µL | Assay Buffer |
| TS | 50 µL | Test Sample |
- Prepare GSH standards (SD), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Note: Treat cells or tissue samples as desired.
- Add 50 µL of GSH working solution into each well of GSH standard, blank control, and test samples to make the total assay volume 100 µL/well. For a 384-well plate, add 25 µL of GSH working solution into each well instead, for total volume of 50 µL/well.
- Incubate the reaction at room temperature for 10 to 60 minutes, protected from light.
- Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 490/525 nm (Cutoff = 515 nm).
Citations
View all 2 citations: Citation Explorer
Xylitol acts as an anticancer monosaccharide to induce selective cancer death via regulation of the glutathione level
Authors: Tomonobu, Nahoko and Komalasari, Ni Luh Gede Yoni and Sumardika, I Wayan and Jiang, Fan and Chen, Youyi and Yamamoto, Ken-ichi and Kinoshita, Rie and Murata, Hitoshi and Inoue, Yusuke and Sakaguchi, Masakiyo
Journal: Chemico-Biological Interactions (2020): 109085
Authors: Tomonobu, Nahoko and Komalasari, Ni Luh Gede Yoni and Sumardika, I Wayan and Jiang, Fan and Chen, Youyi and Yamamoto, Ken-ichi and Kinoshita, Rie and Murata, Hitoshi and Inoue, Yusuke and Sakaguchi, Masakiyo
Journal: Chemico-Biological Interactions (2020): 109085
Comparison of different methods to study effects of silver nanoparticles on the pro-and antioxidant status of human keratinocytes and fibroblasts
Authors: Ahlberg, Sebastian and Rancan, Fiorenza and Epple, Matthias and Loza, Kateryna and Höppe, David and Lademann, Jürgen and Vogt, Annika and Kleuser, Burkhard and Gerecke, Christian and Meinke, Martina C
Journal: Methods (2016)
Authors: Ahlberg, Sebastian and Rancan, Fiorenza and Epple, Matthias and Loza, Kateryna and Höppe, David and Lademann, Jürgen and Vogt, Annika and Kleuser, Burkhard and Gerecke, Christian and Meinke, Martina C
Journal: Methods (2016)
References
View all 131 references: Citation Explorer
Effects of methodological variation on assessment of riboflavin status using the erythrocyte glutathione reductase activation coefficient assay
Authors: Hill MH, Bradley A, Mushtaq S, Williams EA, Powers HJ.
Journal: Br J Nutr (2009): 273
Authors: Hill MH, Bradley A, Mushtaq S, Williams EA, Powers HJ.
Journal: Br J Nutr (2009): 273
A highly sensitive fluorescence probe for fast thiol-quantification assay of glutathione reductase
Authors: Yi L, Li H, Sun L, Liu L, Zhang C, Xi Z.
Journal: Angew Chem Int Ed Engl (2009): 4034
Authors: Yi L, Li H, Sun L, Liu L, Zhang C, Xi Z.
Journal: Angew Chem Int Ed Engl (2009): 4034
A 1,536-well-based kinetic HTS assay for inhibitors of Schistosoma mansoni thioredoxin glutathione reductase
Authors: Lea WA, Jadhav A, Rai G, Sayed AA, Cass CL, Inglese J, Williams DL, Austin CP, Simeonov A.
Journal: Assay Drug Dev Technol (2008): 551
Authors: Lea WA, Jadhav A, Rai G, Sayed AA, Cass CL, Inglese J, Williams DL, Austin CP, Simeonov A.
Journal: Assay Drug Dev Technol (2008): 551
A high-throughput reporter gene assay to prove the ability of natural compounds to modulate glutathione peroxidase, superoxide dismutase and catalase gene promoters in V79 cells
Authors: Ullmann K, Wiencierz AM, Muller C, Thierbach R, Steege A, Toyokuni S, Steinberg P.
Journal: Free Radic Res (2008): 746
Authors: Ullmann K, Wiencierz AM, Muller C, Thierbach R, Steege A, Toyokuni S, Steinberg P.
Journal: Free Radic Res (2008): 746
Assay of glutathione in must and wines using capillary electrophoresis and laser-induced fluorescence detection. Changes in concentration in dry white wines during alcoholic fermentation and aging
Authors: Lavigne V, Pons A, Dubourdieu D.
Journal: J Chromatogr A (2007): 130
Authors: Lavigne V, Pons A, Dubourdieu D.
Journal: J Chromatogr A (2007): 130
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