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Cell Meter™ Intracellular GSH Assay Kit *Optimized for Flow Cytometry*

The decrease in the fluorescence intensity of Thiolite™ Green with the addition of camptothecin in Jurkat cells. Jurkat cells were treated overnight without (blue line) or with 20 µM camptothecin (red line) in a 37 °C, 5% CO2 incubator, and then dye loaded with Thiolite™ Green for 30 minutes. The fluorescence intensity of Thiolite™ Green was measured with a FACSCalibur (Becton Dickinson) flow cytometer using the FL1 channel.
The decrease in the fluorescence intensity of Thiolite™ Green with the addition of camptothecin in Jurkat cells. Jurkat cells were treated overnight without (blue line) or with 20 µM camptothecin (red line) in a 37 °C, 5% CO2 incubator, and then dye loaded with Thiolite™ Green for 30 minutes. The fluorescence intensity of Thiolite™ Green was measured with a FACSCalibur (Becton Dickinson) flow cytometer using the FL1 channel.
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Catalog Number22810
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Excitation (nm)510
Emission (nm)525
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Excitation (nm)
510
Emission (nm)
525
Our Cell Meter™ assay kits are a set of tools for monitoring cell viability. There are a variety of parameters that can be used for monitoring cell viability. This particular kit is designed to monitor cell apoptosis through measuring the depleted antioxidant reduced glutathione (GSH). GSH is involved in many cellular processes including the scavenging of free radicals, drug detoxification, cell signaling, and cell proliferation. The decrease in cellular GSH concentration is an early hallmark in the progression of cell death in response to different apoptotic stimuli in many cell types. Our Cell Meter™ Intracellular GSH Assay Kit provides all the essential components with an optimized assay method for the detection of apoptosis in cells with the decreased GSH. This fluorometric assay is based on the detection of the GSH in cells using our proprietary non-fluorescent Thiolite™ Green dye that becomes strongly fluorescent upon reacting with thiol. In normal cells, Thiolite™ Green accumulates primarily in cytosol, but it is partially translocated to mitochondria in apoptotic cells while Thiolite™ Green staining intensity decreases. Cells stained with Thiolite™ Green can be visualized by flow cytometry. The kit can be paired with other reagents, such as 7-AAD (Cat# 17501), propidium iodide (Cat# 17517) for multi-parametric study of cell viability and apoptosis. The kit is optimized for screening of apoptosis activators and inhibitors by flow cytometry.

Platform


Flow cytometer

Excitation488 nm laser
Emission530/30 nm filter
Instrument specification(s)FITC channel

Components


Component A: Thiolite™ Green1 vial
Component B: Assay Buffer1 bottle (100 mL)
Component C: DMSO1 vial (500 µL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds at a density of 5 × 105 to 1 × 106 cells/mL
  2. Add 5 µL of 200X Thiolite™ Green into 1 mL of cell solution
  3. Incubate the cells in a 37°C, 5% CO2 incubator for 15 to 30 minutes
  4. Pellet the cells and resuspend the cells in 1 mL of growth medium
  5. Analyze cells using flow cytometer with FL1 channel

Important notes
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

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.

1. Thiolite™ Green stock solution (200X):
Add 500 µL of DMSO (Component C) into the vial of Thiolite™ Green (Component A) and mix well to make 200X Thiolite™ Green stock solution. Protect from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

  1. For each sample, prepare cells in 1 mL warm medium or buffer of your choice at a density of 5×105 to 1×106 cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.

  2. Treat cells with test compounds for a desired period of time to induce apoptosis.

  3. Add 5 µL of 200X Thiolite™ Green stock solution into the treated cells.

  4. Incubate the cells in a 37°C, 5% CO2 incubator for 15 to 30 minutes. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact and wash the cells once with serum-containing media prior to the incubation with Thiolite™ Green dye-loading solution. The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.

  5. Optional: Centrifuge the cells at 1000 rpm for 4 minutes and then re-suspend cells in 1 mL of Assay Buffer (Component B) or buffer of your choice.

  6. Monitor the fluorescence intensity using a flow cytometer with FL1 channel (Ex/Em = 490/525 nm). Gate on the cells of interest, excluding debris.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)510
Emission (nm)525

Citations


View all 7 citations: Citation Explorer
The Combination of Astragalus membranaceus and Angelica sinensis Inhibits Lung Cancer and Cachexia through Its Immunomodulatory Function
Authors: Wu, Tsung-Han and Yeh, Kun-Yun and Wang, Chen-Hsu and Wang, Hang and Li, Tsung-Lin and Chan, Yi-Lin and Wu, Chang-Jer
Journal: Journal of oncology (2019)
ROS production and glutathione response in keratinocytes after application of β-carotene and VIS/NIR irradiation
Authors: Lohan, Silke B and Vitt, Kristina and Scholz, Patrik and Keck, Cornelia M and Meinke, Martina C
Journal: Chemico-biological interactions (2017)
Osmotic stress is accompanied by protein glycation in Arabidopsis thaliana
Authors: Paudel, Gagan and Bilova, Tatiana and Schmidt, Rico and Greifenhagen, Uta and Berger, Robert and Tarakhovskaya, Elena and Stöckhardt, Stefanie and Balcke, Gerd Ulrich and Humbeck, Klaus and Br, undefined and t, Wolfgang and others, undefined
Journal: Journal of Experimental Botany (2016): 6283--6295
Systemic Induction of NO-, Redox-, and cGMP Signaling in the Pumpkin Extrafascicular Phloem upon Local Leaf Wounding
Authors: Gaupels, Frank and Furch, Alex and ra CU , undefined and Zimmermann, Matthias R and Chen, Faxing and Kaever, Volkhard and Buhtz, Anja and Kehr, Julia and Sarioglu, Hakan and Kogel, Karl-Heinz and Durner, Jörg
Journal: Frontiers in plant science (2016)
Prediction of intracellular metabolic states from extracellular metabolomic data
Authors: Aurich, Maike K and Paglia, Giuseppe and Rolfsson, Ottar and Hrafnsdóttir, Sigrún and Magnúsdóttir, Manuela and Stefaniak, Magdalena M and Palsson, Bernhard O and Fleming, Ronan MT and Thiele, Ines
Journal: Metabolomics (2015): 603--619
Molecular mechanisms of hyperthermia-induced apoptosis enhanced by withaferin A
Authors: Cui, Zheng-Guo and Piao, Jin-Lan and Rehman, Mati UR and Ogawa, Ryohei and Li, Peng and Zhao, Qing-Li and Kondo, Takashi and Inadera, Hidekuni
Journal: European journal of pharmacology (2014): 99--107
Cancer & Metabolism
Authors: LaMonte, Gregory and Tang, Xiaohu and Chen, Julia Ling-Yu and Wu, Jianli and Ding, Chien-Kuang Cornelia and Keenan, Melissa M and Sangokoya, Carolyn and Kung, Hsiu-Ni and Ilkayeva, Olga and Boros, László G and others, undefined
Journal: (2013)

References


View all 48 references: Citation Explorer
Investigation of the spatial distribution of glutathione redox-balance in live cells by using Fluorescence Ratio Imaging Microscopy
Authors: Maulucci G, Pani G, Labate V, Mele M, Panieri E, Papi M, Arcovito G, Galeotti T, De Spirito M.
Journal: Biosens Bioelectron (2009): 682
Fast determination of glutathione by capillary electrophoresis with fluorescence detection using beta-cyclodextrin as modifier
Authors: Zhang LY, Sun MX.
Journal: J Chromatogr B Analyt Technol Biomed Life Sci (2009): 4051
Determination of free and protein-bound glutathione in HepG2 cells using capillary electrophoresis with laser-induced fluorescence detection
Authors: Wang Y, Xie Y, Bernier M, Wainer IW.
Journal: J Chromatogr A (2009): 3533
Second-order calibration of excitation-emission matrix fluorescence spectra for determination of glutathione in human plasma
Authors: Hemmateenejad B, Rezaei Z, Zaeri S.
Journal: Talanta (2009): 648
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
PARAFAC analysis of the quenching of EEM of fluorescence of glutathione capped CdTe quantum dots by Pb(II)
Authors: Goncalves H, Mendonca C, Esteves da Silva JC.
Journal: J Fluoresc (2009): 141
Fluorescence imaging of cellular glutathione using a latent rhodamine
Authors: Pires MM, Chmielewski J.
Journal: Org Lett (2008): 837
Preparation and characterization of highly fluorescent, glutathione-coated near infrared quantum dots for in vivo fluorescence imaging
Authors: Jin T, Fujii F, Komai Y, Seki J, Seiyama A, Yoshioka Y.
Journal: Int J Mol Sci (2008): 2044
Sensitive and selective determination of glutathione in probiotic bacteria by capillary electrophoresis-laser induced fluorescence
Authors: Musenga A, M and rioli R, Bonifazi P, Kenndler E, Pompei A, Raggi MA.
Journal: Anal Bioanal Chem (2007): 917
High-throughput determination of glutathione and reactive oxygen species in single cells based on fluorescence images in a microchannel
Authors: Gao N, Li L, Shi Z, Zhang X, Jin W.
Journal: Electrophoresis (2007): 3966