ThiolTrace™ Violet 500

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	557.01
Solvent	DMSO

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
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200

Overview SDS Protocol

Platform

Flow cytometer

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

Fluorescence microscope

Excitation	405 nm
Emission	525 nm
Recommended plate	Black wall/clear bottom
Instrument specification(s)	TRITC filterset

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 Note

Thaw at room temperature before starting the experiment.
Note For flow cytometry and fluorescence microscopy, 200 and 500 tests can be performed with the quantity provided, respectively.

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

ThiolTrace^TM Violet 500 stock solution (500X)

Add 200 µL of DMSO (Not provided) 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 1 µL of ThiolTrace^TM Violet 500 stock solution into 0.5 mL of buffer of your choice, and mix well.
Note ThiolTraceTM Violet 500 working solution can be prepared in the cell culture medium without serum.

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 0.5 mL ThiolTrace^TM Violet 500 working solution and incubate them at 37^oC incubator for 20 to 30 minutes.

Note For the fluorescence microscopy, add 200 µL of the ThiolTrace^TM Violet 500 working solution per well.
Centrifuge the cells at 1000 rpm for 4 minutes, and then wash cells in 1 mL of buffer of your choice (Optional).
Resuspend in buffer and 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.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of ThiolTrace™ Violet 500 to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	179.53 µL	897.65 µL	1.795 mL	8.976 mL	17.953 mL
5 mM	35.906 µL	179.53 µL	359.06 µL	1.795 mL	3.591 mL
10 mM	17.953 µL	89.765 µL	179.53 µL	897.65 µL	1.795 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	415
Emission (nm)	499

Product Family

Name	Excitation (nm)	Emission (nm)
CytoCalcein™ Violet 500 Excited at 405 nm	420	505
CytoTell™ Violet 500	415	499

Images

Figure 1. The comparison in the fluorescence intensity of ThiolTrace™ Violet 500 with ThiolTracker^TM Violet (Thermo Scientific) in Jurkat cells in the presence of cell culture medium. Jurkat cells were dye loaded with ThiolTrace™ Violet 500 or ThiolTracker^TM Violet for 20 minutes in a 37 ^oC, 5% CO₂ incubator. The fluorescence intensity was measured using ACEA NovoCyte 3000 flow cytometer with Pacific Orange channel.

Citations

View all 12 citations: Citation Explorer

A highly sensitive two-photon fluorescent probe for glutathione with near-infrared emission at 719nm and intracellular glutathione imaging
Authors: Huang, C., Qian, Y.
Journal: Spectrochim Acta A Mol Biomol Spectrosc (2019): 68-76

Glutathione-driven Cu(i)-O2 chemistry: a new light-up fluorescent assay for intracellular glutathione
Authors: Gao, P. F., Mao, Y. T., Yang, T., Zou, H. Y., Li, Y. F., Huang, C. Z.
Journal: Analyst (2018): 2486-2490

Nitrogen-doped carbon quantum dots as a fluorescent probe to detect copper ions, glutathione, and intracellular pH
Authors: Liao, S., Huang, X., Yang, H., Chen, X.
Journal: Anal Bioanal Chem (2018): 7701-7710

Graphene Quantum Dot-MnO2 Nanosheet Based Optical Sensing Platform: A Sensitive Fluorescence "Turn Off-On" Nanosensor for Glutathione Detection and Intracellular Imaging
Authors: Yan, X., Song, Y., Zhu, C., Song, J., Du, D., Su, X., Lin, Y.
Journal: ACS Appl Mater Interfaces (2016): 21990-6

A sensitive turn-on fluorescent probe for intracellular imaging of glutathione using single-layer MnO2 nanosheet-quenched fluorescent carbon quantum dots
Authors: He, D., Yang, X., He, X., Wang, K., Yang, X., He, X., Zou, Z.
Journal: Chem Commun (Camb) (2015): 14764-7

A mitochondria-targetable fluorescent probe for dual-channel NO imaging assisted by intracellular cysteine and glutathione
Authors: Sun, Y. Q., Liu, J., Zhang, H., Huo, Y., Lv, X., Shi, Y., Guo, W.
Journal: J Am Chem Soc (2014): 12520-3

A fluorescent probe for intracellular cysteine overcoming the interference by glutathione
Authors: Tian, M., Guo, F., Sun, Y., Zhang, W., Miao, F., Liu, Y., Song, G., Ho, C. L., Yu, X., Sun, J. Z., Wong, W. Y.
Journal: Org Biomol Chem (2014): 6128-33

Designing an intracellular fluorescent probe for glutathione: two modulation sites for selective signal transduction
Authors: Isik, M., Guliyev, R., Kolemen, S., Altay, Y., Senturk, B., Tekinay, T., Akkaya, E. U.
Journal: Org Lett (2014): 3260-3

Turn-on fluorescence sensor for intracellular imaging of glutathione using g-C(3)N(4) nanosheet-MnO(2) sandwich nanocomposite
Authors: Zhang, X. L., Zheng, C., Guo, S. S., Li, J., Yang, H. H., Chen, G.
Journal: Anal Chem (2014): 3426-34

Detection of intracellular glutathione using ThiolTracker violet stain and fluorescence microscopy
Authors: M, undefined and avilli, B. S., Janes, M. S.
Journal: Curr Protoc Cytom (2010): Unit 9 35