Live or Dead™ Yeast CFDA-AM/Propidium Iodide Vitality Kit

1. Prepare yeast samples
2. Add 1 µL CFDA-AM dye working solution
3. Add 2 µL propidium iodide dye
4. Vortex gently, and incubate at 37 °C for 15-30 minutes
5. Analyze with flow cytometer using 530/30 nm and 576/26 nm filters

Allow the components to warm to room temperature before opening the vials. Propidium iodide binds to nucleic acids, it should be treated as a potential mutagen and handled with appropriate care. The DMSO stock solution should be handled with particular caution as DMSO is known to facilitate the entry of organic molecules into tissues.

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

1. Add 100 µL of DMSO to the vial of CFDA-AM (Component A). Vortex to mix.
   
   Note        Store stock solution in -20°C in single use aliquots. Avoid repeated freeze-thaws.

The following protocol should only be used as a guideline.

1. Prepare yeast samples containing 1 mL of suspension at ~10⁶ cells/mL.
2. Add 1 µL of CFDA-AM dye working solution and 2 µL of propidium iodide (Component B) to each tube of experimental samples. For unstained controls, place 1 set of tubes aside without adding dye. For single color CFDA-AM controls, add 1 µL of CFDA-AM to one tube of untreated cells and to one tube of killed cells. For single color propidium iodide controls, add 1 µL of propidium iodide to one tube of untreated cells and to one tube of killed cells. Please note, depending upon your specific application the proportion of the two dyes may need to be adjusted for optimal response.
3. Vortex each tube gently. Then incubate all samples at room temperature for 15 to 30 minutes, protected from light.
4. Monitor the fluorescence increase using a flow cytometer equipped with a 488 nm laser, and a 530/30 nm filter and a 575/26 nm filter (FITC and PE channel).
   
   Note        To analyze the samples with fluorescence microscopy, trap 5 µL of the stained yeast suspension between a slide and a coverslip. Observe in a microscope using FITC and Cy3 filter set.