ThiolTrace™ Violet 500

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

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.

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

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

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.

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.

1. 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.
2. Centrifuge the cells at 1000 rpm for 4 minutes, and wash cells in 1 mL of buffer of your choice (Optional).
3. 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.
4. Centrifuge the cells at 1000 rpm for 4 minutes, and then wash cells in 1 mL of buffer of your choice (Optional).
5. 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.