Two-parameter cell health assay
This kit enables simultaneous determination of live and dead cells with two probes: CellTrace™ calcein violet indicates cell viability based on plasma membrane integrity, and aqua-fluorescent reactive dye measures cell vitality based on intracellular esterase activity.
This protocol can be used for:
- Identifying live and dead cells using a flow cytometer
This protocol should not be used for:
- Fluorescence microscopy
|1. Thaw one vial each of components A, B, and C|
|2. Add 50 μL DMSO (Component C) to one vial of aqua-fluorescent reactive dye (Component B)|
|3. Add 42 μL DMSO (Component C) to one vial of calcein violet AM (Component A) to prepare a stock solution|
|4. Add 40 μL of this stock solution to 1.25 mL of buffer or medium to make a working solution of calcein violet AM|
|5. Add 1 mL cells to a flow cytometer tube. Cells may be suspended in serum-free medium or buffer|
|6. Add 1 μL aqua-fluorescent reactive dye working solution and 5 μL calcein violet AM working solution to each mL cell suspension|
|7. Mix the sample|
|8. Incubate the cells for 30 minutes at room temperature or on ice|
|9. Wash once and resuspend in buffer|
|10. Run cells on a flow cytometer using violet (~405 nm) excitation and violet fluorescence emission (~450 nm) for the calcein violet (live cells) and blue-green fluorescence emission (~525 nm) for the aqua-fluorescent reactive dye (dead cells); minimal compensation will be necessary|
- Cell concentration should be 1 x 105 to 5 x 106 cells per mL
- Stock solutions of both dyes should preferably be used within 24 hours
- Calcein violet AM may hydrolyze if exposed to moisture
- Not compatible with fixation
Staining pattern of a mixture of heat-killed and untreated Jurkat cells (human leukemia T-cell) stained with the LIVE/DEAD® Violet Viability/Vitality Kit.
|Aqua-fluorescent reactive dye||Calcein violet AM|
|Excitation/Emission (in nm)||367/526||400/452|
For Research Use Only. Not for use in diagnostic procedures.