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1. Prepare 10 mM DMSO stock solution

For #22022: Dissolve 5.6 mg in 1 ml DMSO to make 10 mM stock solution (1 mg/ml is equivalent to 1.8 mM); For #22028 add 90 µL DMSO to each vial to make 10 mM stock solution.

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.

2. Prepare CFSE working solution

Prepare a CFSE working solution (0.5 to 5 µM) right before use by diluting the DMSO stock solution (20,000 to 2,000 times) from Step 1 with Hanks and 20 mM Hepes buffer (HHBS) or the buffer of your choice, pH 7. Mix them well by vortexing.

3. Analyze cells with a flow cytometer or a fluorescence microscope:

3.1    Treat cells with test compounds for a desired period of time.

3.2    Centrifuge the cells to get 1-5 × 10⁵ cells per tube.

3.3     Resuspend cells in 500 μL of the dye working solution (from Step 2).

3.4     Incubate cells with a dye solution at room temperature or 37 °C for 10 to 30 min, protected from light.

3.5     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.

3.6     Monitor the fluorescence change at Ex/Em = 490/520 nm with a flow cytometer (FL1 channel) or a fluorescence microscope.

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