image of a Molecular Probes product pouch

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

You will need the following for this protocol:

Protocol

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

 

Protocol tips

  • 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


flow cytometry scatter plot showing two distinct populations: live cells (stained with calcein) and dead cells (stained with aqua fluorescent dye)

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.

Spectral information and storage
  Aqua-fluorescent reactive dye Calcein violet AM
Excitation/Emission (in nm) 367/526 400/452
Storage conditions –20°C –20°C