CytoTrace™ Ultra Green

Protocol summary

1. Prepare cells with test compounds
2. Add 0.5 to 5 µM CytoTrace™ Ultra Green working solution
3. Incubate dyes with cells at room temperature or 37 ^oC for 15 to 30 minutes
4. Remove the dye working solution
5. Analyze with a flow cytometer with Ex/Em = 490/520 nm-FITC filter set

CytoTrace™ Ultra Green stock solution (2-10 mM):
Add appropriate amount of DMSO and mix well to make CytoTrace™ Ultra Green stock solution (2-10 mM). 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.

CytoTrace™ Ultra Green working solution:
Prepare a 0.5 to 5 µM dye working solution right before use by diluting the DMSO stock solution from with Hanks and 20 mM Hepes buffer (HHBS) or the buffer of your choice, pH 7. Mix them well by vortexing. Note: In some cell types, lower concentration may be required to stain the cells. We recommend optimizing optimal concentration for each cell type before performing experiment.

1. Treat cells with test compounds for a desired period of time.
   
   
2. Centrifuge the cells to get 2-10 × 10⁵ cells per tube.
   
   
3. Resuspend cells in 500 µL of the CytoTrace™ Ultra Green working solution.
   
   
4. Incubate cells with a dye solution at room temperature or 37 °C for 15 to 30 min, protected from light.
   
   
5. Remove the dye working solution from the cells; Optional: The cells can be fixed with 4% formaldehyde.
   
   
6. Wash the cells with HHBS or buffer of your choice once.
   
   
7. Resuspend cells in 500 µL of pre-warmed HHBS or medium to get 2-10 × 10⁵ cells per tube.
   
   
8. Monitor the fluorescence change at Ex/Em = 490/520 nm with a flow cytometer or a fluorescence microscope with FITC filter set.