Intracellular glutathione (GSH) Detection Assay Kit (ab112132)

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Overview

  • Product name
    Intracellular glutathione (GSH) Detection Assay Kit
    See all Glutathione kits
  • Detection method
    Fluorescent
  • Sample type
    Adherent cells, Suspension cells
  • Product overview

    Intracellular glutathione (GSH) Detection Assay Kit (ab112132) uses our proprietary non-fluorescent Green Dye, which becomes strongly fluorescent upon reacting with thiol (including GSH in cells). In normal cells, the Green Dye is accumulated primarily in cytosol, but it is partially translocated to mitochondria in apoptotic cells while Green Dye staining intensity is decreased. Cells stained with Green Dye can be visualized with a flow cytometer at Ex/Em = 490/520 nm (FL1 channel).
    This kit has been designed to detect apoptosis is cells by measuring the decrease in reduced glutathione (GSH), which is an early hallmark in the progression of cell death in response to different apoptotic stimuli in many cells.

    This kit can be used together with other reagents, such as 7-AAD for multi-parametric study of cell viability and apoptosis. The kit is optimized for screening apoptosis activators and inhibitors with a flow cytometer.

     

    We do not recommend using this kit in a microplate. A more suitable option might be ab65322.

     

    Visit our FAQs page for tips and troubleshooting.

  • Notes

    Glutathione is a tripeptide that contains L-cysteine, L-glutamic acid and glycine. It is the smallest intracellular protein thiol molecule in the cells, which prevents cell damage caused by reactive oxygen species such as free radicals and peroxides. Glutathione exists in reduced (GSH) and oxidized (GSSG) states. Reduced glutathione (GSH) is a major tissue antioxidant that provides reducing equivalents for the glutathione peroxidase (GPx) catalyzed reduction of lipid hydroperoxides to their corresponding alcohols and hydrogen peroxide to water. In the GPx catalyzed reaction, the formation of a disulfide bond between two GSH molecules generates oxidized glutathione (GSSG). The enzyme glutathione reductase (GR) recycles GSSG to GSH with the simultaneous oxidation of β-nicotinamide adenine dinucleotide phosphate (β-NADPH2). In healthy cells, more than 90% of the total glutathione pool is in the reduced form (GSH). When cells are exposed to increased levels of oxidative stress, GSSG accumulates and the ratio of GSSG to GSH increases. An increased ratio of GSSG-to-GSH is an indication of oxidative stress. The monitoring of reduced and oxidized GSH in biological samples is essential for evaluating the redox and detoxification status of the cells and tissues against oxidative and free radicals mediated cell injury.

  • Platform
    Flow cytometer

Properties

Images

  • Intracellular GSH Assay Kit (ab112132)
    Intracellular GSH Assay Kit (ab112132)

    Decrease in the fluorescence intensity of the Thiol Green Dye correlates with the addition of camptothecin in Jurkat cells. Jurkat cells were mock induced with 0.25% DMSO (blue line) and with 12.5 μM camptothecin (black line) in a 37ºC, 5% CO2 incubator for 24 hours and then dye loaded with ab112132 Green Dye for 30 minutes. The fluorescence intensity of Green Dye was measured with a flow cytometer using the FL1 channel.

Protocols

References

This product has been referenced in:
  • Moniruzzaman R  et al. Roles of intracellular and extracellular ROS formation in apoptosis induced by cold atmospheric helium plasma and X-irradiation in the presence of sulfasalazine. Free Radic Biol Med 129:537-547 (2018). Read more (PubMed: 30355525) »
  • Castex J  et al. Inactivation of Lsd1 triggers senescence in trophoblast stem cells by induction of Sirt4. Cell Death Dis 8:e2631 (2017). Read more (PubMed: 28230862) »
See all 5 Publications for this product

Publishing research using ab112132? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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Question

Intracellular GSH Assay Kit (ab112132)Are the cells viable after the assay? Is the Green Dye toxic to the cells or can the cells be collected from the FACS and further cultured?
thanks,

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Answer

The concentration of the dye used in the assay is toxic to cells, so these can not be further cultured.

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Question

Hi,

I am using Greiner Thincert Culture Inserts for 6-well plates and A549 cells in my exposure experiments. The goal is to expose the cells at an air/liquid interphase for a certain period and to measure cell viability, oxidative stress markers and cell cycle stage after the exposure.

Until now I have tried to detach the cells from the inserts and measure the MTT assay and do flow cytometry analysis. However I have been having problems detaching the cells from the inserts, trypsin/EDTA is ineffective and scraping destroys the cells, so I have not been able to get sensible data from our exposure experiments.

Can you recommend any kits/procedures which could be applied directly in the inserts without prior detachments of the cells?

The minimum parameters I would need to evaluate after the exposure are cell viability and apoptosis/cell cycle stage, but I would like to include a marker for oxidative stress (e.g. glutathione) as well. I should be able to measure all the mentioned parameters simultaneously in one insert without too much quenching and fluorescence interference/overlay.

We have a Victor III microplate reader and an inverted fluorescence microscope for which we will acquire quantification software shortly.

Thank you for your help.

With best regards,

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Answer

Thank you for your enquiry and interest in our products.

MTT assay can be carried out on 96 well plates using either adherent or floating cells. You do not necessary need to remove (harvest) the cells by trypsinization. You can culture the cells in the wells of the 96 well plates, then apply the MTT solution (20ul) to each well. During incubation (37C, 5% CO2) for 1-5 hours MTT will be metabolized. The formazan (MTT metabolic product) can then be dissolved in DMSO and the optical density can be read at 560 nm using a plate reader - without harvesting the cells. There may be other cell viability/cytotoxicyprotovols but this is the most widely used.

Cell viability kits:

We have different cell viability assay kits in our catalogue i.e. ab112120, ab112121, ab112122, ab112123.

https://www.abcam.com/Cell-Viability-Assay-Kit-Fluorometric-Blue-ab112120.html

https://www.abcam.com/Cell-Viability-Assay-Kit-Fluorometric-Dual-Green-Red-ab112121.html

https://www.abcam.com/Cell-Viability-Assay-Kit-Fluorometric-Green-ab112122.html

https://www.abcam.com/Cell-Viability-Assay-Kit-Fluorometric-Near-InfraRed-ab112123.html

GSH kits:

GSH can be detected/quantified either by fluorimetric or chromogenic (DTNB - Ellman's reagent) assays and currently we have also some assay kits for detecting this tripeptide, you may wish to take a look at the datasheets for further information.

- ab65322: Glutathione Fluorometric Detection Kit

https://www.abcam.com/Glutathione-Fluorometric-Detection-Kit-ab65322.html

- ab112132:Intracellular GSH Assay Kit

https://www.abcam.com/Intracellular-GSH-Assay-Kit-ab112132.html

Normally, intracellular GSH levels can be expressed per cell numbers or per protein concentration.

I hope this helps and if I can assist further, please do not hesitate to contact me.

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