Live or Dead™ Yeast Viability Assay Kit
Additional ordering information
|Shipping||Standard overnight for United States, inquire for international|
Storage, safety and handling
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
|Storage||Freeze (< -15 °C); Minimize light exposure|
The Live or Dead™ Yeast Viability Assay Kit is a reliable and robust assay that allows researchers to quantitatively distinguish live and dead yeast within minutes using flow cytometry. This kit contains two ready-to-use nucleic acid stains: MycoLight™ Green JJ98, a replacement for SYTO™ 9, and propidium iodide. MycoLight™ Green JJ98 is a green fluorescent nucleic acid stain that labels all yeast populations - live yeast with intact membranes and dead yeast with damaged membranes. In contrast, propidium iodide only stains dead yeast with damaged membranes. In dead yeasts where both dyes are present, the fluorescence of MycoLight™ Green JJ98 is notably reduced due to FRET by propidium iodide. As a result, live yeast with intact membranes will stain fluorescent green, and dead yeast with damaged membranes will stain fluorescent red.
AT A GLANCE
- Prepare yeast samples
- Add 1 µL MycoLight™ Green JJ98 dye
- Add 2 µL propidium iodide dye
- Gently vortex, and incubate at 37°C for 15-30 minutes
- Analyze with flow cytometer using 530/30 nm and 576/26 nm filters
ImportantAllow 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.
SAMPLE EXPERIMENTAL PROTOCOL
The following protocol should be used only as a guideline.
- Prepare yeast samples containing 1 mL of suspension at ~106 cells/mL.
- Add 1 µL of MycoLight™ Green JJ98 (Component A) 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 MycoLight™ Green JJ98 controls, add 1 µL of MycoLight™ Green JJ98 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.
- Vortex each tube gently. Then incubate all samples at room temperature for 15 to 30 minutes, protected from light.
- 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.
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of Live or Dead™ Yeast Viability Assay Kit to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
|0.1 mg||0.5 mg||1 mg||5 mg||10 mg|
|1 mM||149.613 µL||748.066 µL||1.496 mL||7.481 mL||14.961 mL|
|5 mM||29.923 µL||149.613 µL||299.226 µL||1.496 mL||2.992 mL|
|10 mM||14.961 µL||74.807 µL||149.613 µL||748.066 µL||1.496 mL|
Enter any two values (mass, volume, concentration) to calculate the third.
|Mass (Calculate)||Molecular weight||Volume (Calculate)||Concentration (Calculate)||Moles|