Cell Meter™ Intracellular GSH Assay Kit Optimized for Flow Cytometry with 405 nm excitation

The decrease in the fluorescence intensity of ThiolTrace™ Violet 500 with the addition of Campothecin in HL-60 cells. HL-60 cells were treated for 6 hours without (Red line) or with 20 μM Campothecin (Green line) in a 37 <sup>o</sup>C, 5% CO<sub>2</sub> incubator, and then stained with ThiolTrace™ Violet 500 for 20 minutes. The fluorescence intensity was measured using ACEA NovoCyte 3000 flow cytometer with Pacific Orange channel.

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Spectral properties

Excitation (nm)	415
Emission (nm)	499

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Alternative formats

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

Overview SDS Protocol

Excitation (nm)

415

Emission (nm)

499

There are a variety of parameters that can be used for monitoring cell apoptosis. This particular kit is designed to detect cell apoptosis by measuring the decrease in reduced glutathione (GSH). GSH is important for maintaining redox level of cells. It 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 cells. Our Cell Meter™ Intracellular GSH Assay Kit uses our proprietary ThiolTrace™ Violet 500, which becomes strongly fluorescent upon reacting with thiol (including GSH in cells). In normal cells, probe is highly fluorescent, while in apoptotic cells, staining intensity is decreased. Cells stained with ThiolTrace™ Violet can be visualized with a flow cytometer (Pacific Orange filter set). The kit can be used together with other reagents, such as 7-AAD (#17501) for multi-parametric study of cell viability and apoptosis. The kit is optimized for screening apoptosis activators and inhibitors with a flow cytometer.

Platform

Flow cytometer

Excitation	405 nm laser
Emission	525/50 nm filter
Instrument specification(s)	Pacific Orange channel

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare cells with test compounds at a density of 5 × 10⁵ to 1 × 10⁶ cells/mL
Prepare and add ThiolTrace^TM Violet 500 working solution to cells
Incubate at 37^oC for 20 to 30 minutes
Read fluorescence intensity at Ex/Em = 405/525 nm-Pacific Orange filter set

Important notes
Thaw 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.

ThiolTrace^TM Violet 500 stock solution (500X):
Add 200 µL of DMSO (Component C) into the vial of ThiolTrace^TM Violet 500, and mix well. Note: Aliquot and stored the unused ThiolTrace^TM Violet 500 stock solution at -20^oC. Avoid repeated freeze/thaw cycles.

PREPARATION OF WORKING SOLUTION

ThiolTrace^TM Violet 500 working solution (1X):
Add 2 µL of ThiolTrace^TM Violet 500 stock solution into 1 mL of Assay Buffer (Component B) or buffer of your choice, and mix well. Note: ThiolTrace^TM Violet 500 working solution can be prepared in the cell culture medium containing serum.

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

SAMPLE EXPERIMENTAL PROTOCOL

Treat cells with test compounds for a desired period of time. 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 ThiolTrace™ Violet working solution. Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.
Centrifuge the cells at 1000 rpm for 4 minutes, and wash cells in 1 mL of buffer of your choice (Optional).
Resuspend cells in 1 mL ThiolTrace^TM Violet 500 working solution and incubate them at 37^oC incubator for 20 to 30 minutes.
Monitor the fluorescence intensity with a flow cytometer using Pacific Orange filter set (Ex/Em = 405/525 nm). Gate on the cells of interest, excluding debris.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	415
Emission (nm)	499

Images

Figure 1. The decrease in the fluorescence intensity of ThiolTrace™ Violet 500 with the addition of Campothecin in HL-60 cells. HL-60 cells were treated for 6 hours without (Red line) or with 20 μM Campothecin (Green line) in a 37 ^oC, 5% CO₂ incubator, and then stained with ThiolTrace™ Violet 500 for 20 minutes. The fluorescence intensity was measured using ACEA NovoCyte 3000 flow cytometer with Pacific Orange channel.

Citations

View all 50 citations: Citation Explorer

Glutathione S-transferase Pi 1 is a valuable predictor for cancer drug resistance in esophageal squamous cell carcinoma
Authors: Ogino, S., Konishi, H., Ichikawa, D., Matsubara, D., Shoda, K., Arita, T., Kosuga, T., Komatsu, S., Shiozaki, A., Okamoto, K., Kishimoto, M., Otsuji, E.
Journal: Cancer Sci (2019): 795-804

Whole-cell cascade for the preparation of enantiopure beta-O-4 aryl ether compounds with glutathione recycling
Authors: Husarcikova, J., Schallmey, A.
Journal: J Biotechnol (2019): 1-7

Glutathione S-transferases P1 protects breast cancer cell from adriamycin-induced cell death through promoting autophagy
Authors: Dong, X., Yang, Y., Zhou, Y., Bi, X., Zhao, N., Zhang, Z., Li, L., Hang, Q., Zhang, R., Chen, D., Cao, P., Yin, Z., Luo, L.
Journal: Cell Death Differ (2019): se name="22809.enl" path="C:\Users\aatbi\Drop

Mitochondria-Accumulating Rhenium(I) Tricarbonyl Complexes Induce Cell Death via Irreversible Oxidative Stress and Glutathione Metabolism Disturbance
Authors: Wang, F. X., Liang, J. H., Zhang, H., Wang, Z. H., Wan, Q., Tan, C. P., Ji, L. N., Mao, Z. W.
Journal: ACS Appl Mater Interfaces (2019): 13123-13133

Evaluation of Glutathione S-Transferase Inhibition Effects on Idiopathic Pulmonary Fibrosis Therapy with a Near-Infrared Fluorescent Probe in Cell and Mice Models
Authors: He, N., Bai, S., Huang, Y., Xing, Y., Chen, L., Yu, F., Lv, C.
Journal: Anal Chem (2019): 5424-5432

Dual role of BSA for synthesis of MnO2 nanoparticles and their mediated fluorescent turn-on probe for glutathione determination and cancer cell recognition
Authors: Wang, Q., Zhang, Y., Wang, X., Wu, Y., Dong, C., Shuang, S.
Journal: Analyst (2019): 1988-1994

Deficiency of the mitochondrial sulfide regulator ETHE1 disturbs cell growth, glutathione level and causes proteome alterations outside mitochondria
Authors: Sahebekhtiari, N., Fern and ez-Guerra, P., Nochi, Z., Carlsen, J., Bross, P., Palmfeldt, J.
Journal: Biochim Biophys Acta Mol Basis Dis (2019): 126-135

Glutathione S-transferase pi 1 variant and squamous cell carcinoma susceptibility: a meta-analysis of 52 case-control studies
Authors: Wang, S., Zhang, J., Jun, F., Bai, Z.
Journal: BMC Med Genet (2019): 22

Carbon monoxide sensitizes cisplatin-resistant ovarian cancer cell lines toward cisplatin via attenuation of levels of glutathione and nuclear metallothionein
Authors: Kawahara, B., Ramadoss, S., Chaudhuri, G., Janzen, C., Sen, S., Mascharak, P. K.
Journal: J Inorg Biochem (2019): 29-39

Glutathione S-transferase theta genotypes and environmental exposures in the risk of canine transitional cell carcinoma
Authors: Luethcke, K. R., Ekena, J., Chun, R., Trepanier, L. A.
Journal: J Vet Intern Med (2019): ersion="1.0" encoding="UTF-8" ?>22809.enlEndN