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Thiolite™ Blue, AM
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.
Example protocol
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.
Thiolite™ Blue AM Stock Solution
Prepare a 1 to 5 mM stock solution of Thiolite™ Blue in high-quality, anhydrous DMSO.PREPARATION OF WORKING SOLUTION
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.
SAMPLE EXPERIMENTAL PROTOCOL
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.
- Prepare viable cells as desired.
- Treat cells with test compounds.
- Centrifuge the cells to get 1-5 × 105 cells per tube.
- 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 - Incubate the dye-loaded plate in a cell incubator at 37 °C for 15 to 60 minutes.
- Remove the dye working solution and wash cells with HHBS or buffer of your choice to remove any excess probes.
- Resuspend the cells in 1 mL of pre-warmed HHBS or medium to get 2-10 × 105 cells per tube.
- 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.
Calculators
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 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 301.859 µL | 1.509 mL | 3.019 mL | 15.093 mL | 30.186 mL |
| 5 mM | 60.372 µL | 301.859 µL | 603.719 µL | 3.019 mL | 6.037 mL |
| 10 mM | 30.186 µL | 150.93 µL | 301.859 µL | 1.509 mL | 3.019 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
Product family
| Name | Excitation (nm) | Emission (nm) |
| Calcein Blue, AM *CAS 168482-84-6* | 354 | 441 |
Citations
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
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
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
References
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
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)
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
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
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
Authors: Guo XF, Zhao PX, Wang H, Zhang HS.
Journal: J Chromatogr B Analyt Technol Biomed Life Sci (2011): 3932
Page updated on November 21, 2024
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