Overview

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1. Prepare 500 X DMSO stock solution

Add 500 µL DMSO into the dye powder vial, mix it well by vortexing to have a 500X DMSO 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 1X dye working solution

Prepare a 1X dye working solution by diluting 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 a t en fold range. Such as CytoTell™ Red might use much less amount in some cell types than the recommend concentrations.

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).

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)

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 respected Ex/Em (see Table 1) with a flow cytometer or a fluorescence microscope.

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