Calculator
CytoTell™ Violet 500
Example protocol
AT A GLANCE
- Prepare cells with test compounds
- Add 1X dye working solution
- Incubate dyes with cells at room temperature or 37 oC for 10 to 30 minutes
- Remove the dye working solution
- Analyse with flow cytometer with appropriate filter set
Bring all the kit components at room temperature before starting the experiment.
Note: The CytoTell™ dyes are lyophilized powders. They should be stable for at least 6 months if store at -20 °C, protecting from light, and avoiding freeze/thaw cycles.
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
Add 500 µL DMSO into the dye powder vial, mix it well by vortexing to have a stock solution (500X).
Note: The stock solution should be used promptly; any remaining solution should be aliquoted and frozen at < - 20 oC. Avoid repeated freeze-thaw cycles, and protect from light.
PREPARATION OF WORKING SOLUTION
Dilute the 500X DMSO stock solution at 1 to 500 in Hanks and 20 mM Hepes buffer (HHBS) or the buffer of your choice, pH 7 (such as 1 µL of 500X DMSO stock solution to 500 µL buffer) right before use. 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 at the concentrations that are at least over ten fold range. Such as CytoTell™ Red might use much less amount in some cell types than the recommend concentrations.
SAMPLE EXPERIMENTAL PROTOCOL
- Treat cells with test compounds for a desired period of time.
- Centrifuge the cells to get 1-5 × 105 cells per tube.
Resuspend cells in 500 µL of the CytoTell™ dye working solution.
Optional: One can add the 500X DMSO stock solution into the cells directly without medium removing (such as, add 1 µL500X DMSO stock solution into 500 µL cells)
- Incubate cells with a dye solution at room temperature or 37 °C for 10 to 30 minutes, protected from light.
- Remove the dye working solution from the cells, wash the cells with HHBS or buffer of your choice. Resuspend cells in 500 µL of pre-warmed HHBS or medium to get 1-5 × 105 cells per tube.
- Monitor the fluorescence change at respected Ex/Em (see Table 1) with a flow cytometer or a fluorescence microscope.
Calculators
Common stock solution preparation
| 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
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) |
| CytoCalcein™ Violet 500 *Excited at 405 nm* | 420 | 505 |
| ThiolTrace™ Violet 500 | 415 | 499 |
Citations
Authors: Alvarez, R., Jr., Wang, B. J., Quijada, P. J., Avitabile, D., Ho, T., Shaitrit, M., Chavarria, M., Firouzi, F., Ebeid, D., Monsanto, M. M., Navarrete, N., Moshref, M., Siddiqi, S., Broughton, K. M., Bailey, B. A., Gude, N. A., Sussman, M. A.
Journal: J Mol Cell Cardiol (2019): 154-164
Authors: Jun, E. J., Song, H. Y., Park, J. H., Bae, Y. S., Paulson, B., Lee, S., Cho, Y. C., Tsauo, J., Kim, M. T., Kim, K. Y., Yang, S. G., Kim, J. K.
Journal: J Vasc Interv Radiol (2018): 1756-1763
Authors: Luan, M., Yu, L., Li, Y., Pan, W., Gao, X., Wan, X., Li, N., Tang, B.
Journal: Anal Chem (2017): 10601-10607
Authors: Zolnierowicz, J., Ambrozek-Latecka, M., Kawiak, J., Wasilewska, D., Hoser, G.
Journal: Folia Histochem Cytobiol (2013): 193-200
Authors: Begum, J., Day, W., Henderson, C., Purewal, S., Cerveira, J., Summers, H., Rees, P., Davies, D., Filby, A.
Journal: Cytometry A (2013): 1085-95
Safety data sheet