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Thiolite™ Blue, AM

The excitation and emission spectra of Thiolite<sup>TM</sup> Blue, AM.
The excitation and emission spectra of Thiolite<sup>TM</sup> Blue, AM.
The excitation and emission spectra of Thiolite<sup>TM</sup> Blue, AM.
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Physical properties
Molecular weight331.28
Spectral properties
Excitation (nm)335
Emission (nm)460
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure


Molecular weight
Excitation (nm)
Emission (nm)
Thiolite™ Blue is one of the most sensitive sensors for measuring thiol compounds. It gives a blue fluorescent adduct upon reacting with thiol compounds (such as GSH and cysteine). It can be used to quantifying the number of cysteines on a protein. We have used it to measure glutathione fluorimetrically. It has >200-fold fluorescence enhancement upon reaction with thiol-containing compounds. Thiolite™ Blue is an excellent replacement for mBBr (monobromobimane) due to their similar spectral properties. Compared to mBBr, the thiol adduct of Thiolite™ Blue has much stonger fluorescence and absorption than that of mBBr, making it a much more sensitive thiol probe than bromobimanes. Thiolite™ Blue is optimized for intracellular thiol detection. It is non-fluorescent outside cells, eliminating the wash step and reducing assay background.


Flow cytometer

Excitation350 nm or 405 nm laser
Emission450/40 nm filter
Instrument specification(s)Pacific Blue channel

Fluorescence microscope

ExcitationDAPI filter set
EmissionDAPI filter set
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Example protocol


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.

Thiolite™ Blue AM Stock Solution
Prepare a 1 to 5 mM stock solution of Thiolite™ Blue in high-quality, anhydrous DMSO.


Thiolite™ Blue AM Working Solution
On the day of the experiment, either dissolve Thiolite™ Blue AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a 1X Thiolite™ Blue AM working solution in a buffer of your choice (e.g., Hanks and Hepes buffer, pH 7), and mix them well by vortexing.
Note     The final concentration of the dye working solution should be empirically determined for different cell types and/or experimental conditions. It is recommended to test concentrations that are at least over a ten-fold range. The recommended concentration in Jurkat cells is 1-10 µM.


The following is a recommended protocol for loading Thiolite™ Blue AM into live mammalian cells. This protocol only provides a guideline, should be modified according to your specific needs.
  1. Prepare viable cells as desired.
  2. Treat cells with test compounds.
  3. Centrifuge the cells to get 1-5 × 105 cells per tube.
  4. Resuspend cells in 1 mL of Thiolite™ Blue AM working solution.
    Note     Alternatively, the DMSO stock solution can be added directly to the cells without removing the medium. For example, add 1 µL of 1 mM DMSO stock solution into 1 mL cells
  5. Incubate the dye-loaded plate in a cell incubator at 37 °C for 15 to 60 minutes.
  6. Remove the dye working solution and wash cells with HHBS or buffer of your choice to remove any excess probes.
  7. Resuspend the cells in 1 mL of pre-warmed HHBS or medium to get 2-10 × 105 cells per tube.
  8. Measure the fluorescence intensity using either a fluorescence microscope equipped with a DAPI filter set, a flow cytometer equipped with a UV/violet laser and a 450/40 nm filter (Pacific Blue channel), or a fluorescence plate reader at Ex/Em = 340/460 nm cutoff 420 nm. 


Common stock solution preparation

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

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM301.859 µL1.509 mL3.019 mL15.093 mL30.186 mL
5 mM60.372 µL301.859 µL603.719 µL3.019 mL6.037 mL
10 mM30.186 µL150.93 µL301.859 µL1.509 mL3.019 mL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles


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Spectral properties

Excitation (nm)335
Emission (nm)460

Product Family

NameExcitation (nm)Emission (nm)
Calcein Blue, AM *CAS 168482-84-6*354441



View all 2 citations: Citation Explorer
Gas Chromatography/Mass Spectrometry-Based Metabolomic Profiling Reveals Alterations in Mouse Plasma and Liver in Response to Fava Beans
Authors: Xiao, Man and Du, Guankui and Zhong, Guobing and Yan, Dongjing and Zeng, Huazong and Cai, Wangwei
Journal: PloS one (2016): e0151103
Virus-like particles with removable cyclodextrins enable glutathione-triggered drug release in cells
Authors: Niikura, Kenichi and Sugimura, Naotoshi and Musashi, Yusuke and Mikuni, Shintaro and Matsuo, Yasutaka and Kobayashi, Shintaro and Nagakawa, Keita and Takahara, Shuko and Takeuchi, Chie and Sawa, Hirofumi and others, undefined
Journal: Molecular biosystems (2013): 501--507


View all 74 references: Citation Explorer
Thiol-yne reaction on boron-doped diamond electrodes: application for the electrochemical detection of DNA-DNA hybridization events
Authors: Meziane D, Barras A, Kromka A, Houdkova J, Boukherroub R, Szunerits S.
Journal: Anal Chem (2012): 194
Simultaneous Nucleophilic-substituted and Electrostatic Interactions for Thermal Switching of Spiropyran: A New Approach for Rapid and Selective Colorimetric Detection of Thiol-containing Amino Acids
Authors: Li Y, Duan Y, Li J, Zheng J, Yu H, Yang R.
Journal: Anal Chem. (2012)
A sensitive and rapid ultra HPLC-MS/MS method for the simultaneous detection of clopidogrel and its derivatized active thiol metabolite in human plasma
Authors: Peer CJ, Spencer SD, VanDenBerg DA, Pacanowski MA, Horenstein RB, Figg WD.
Journal: J Chromatogr B Analyt Technol Biomed Life Sci (2012): 132
Investigation of chemically modified barium titanate beads as surface-enhanced Raman scattering (SERS) active substrates for the detection of benzene thiol, 1,2-benzene dithiol, and rhodamine 6G
Authors: Onuegbu J, Fu A, Glembocki O, Pokes S, Alexson D, Hosten CM.
Journal: Spectrochim Acta A Mol Biomol Spectrosc (2011): 456
Simple and rapid determination of thiol compounds by HPLC and fluorescence detection with 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide) difluoroboradiaza-s-indacene
Authors: Guo XF, Zhao PX, Wang H, Zhang HS.
Journal: J Chromatogr B Analyt Technol Biomed Life Sci (2011): 3932
Selective chromogenic detection of thiol-containing biomolecules using carbonaceous nanospheres loaded with silver nanoparticles as carrier
Authors: Hu B, Zhao Y, Zhu HZ, Yu SH.
Journal: ACS Nano (2011): 3166
Thiol redox proteomics seen with fluorescent eyes: the detection of cysteine oxidative modifications by fluorescence derivatization and 2-DE
Authors: Izquierdo-Alvarez A, Martinez-Ruiz A.
Journal: J Proteomics (2011): 329
Indicator approach to develop a chemosensor for the colorimetric sensing of thiol-containing water and its application for the thiol detection in plasma
Authors: Huo FJ, Yang YT, Su J, Sun YQ, Yin CX, Yan XX.
Journal: Analyst (2011): 1892
Thiol-exchange in DTSSP crosslinked peptides is proportional to cysteine content and precisely controlled in crosslink detection by two-step LC-MALDI MSMS
Authors: Lambert W, Soderberg CA, Rutsdottir G, Boelens WC, Emanuelsson C.
Journal: Protein Sci (2011): 1682
Modulating DNA-templated silver nanoclusters for fluorescence turn-on detection of thiol compounds
Authors: Huang Z, Pu F, Lin Y, Ren J, Qu X.
Journal: Chem Commun (Camb) (2011): 3487