Thiolite™ Blue, AM

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

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

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.