Two-color dual-parameter cell viability assay

This kit permits quick and easy determination of cell viability using two common microscope filters (FITC and RFP) based on intracellular esterase activity and plasma membrane integrity.

This protocol can be used for:

  • Identifying live and dead cells using a fluorescence microscope

This protocol should not be used for:

  • Flow cytometry

You will need the following for this protocol:



1. Culture cells in appropriate medium and vessel for microscopy


2. Thaw vials


3. Add 5 µL calcein AM (Component A) and 20 µL ethidium homodimer-1 (Component B) to 10 mL DPBS to create staining solution.


4. Remove medium from cells


5. Add 100–200 µL of the staining solution directly to cells


6. Incubate 30 minutes at 20–25°C


7. Image cells

Spectral information and storage

  Calcein AM Ethidium homodimer-1
Excitation/emission 494/517 nm 528/617 nm
Standard filter set FITC or GFP RFP
EVOS® Light Cube GFP RFP
Storage conditions –20°C –20°C


Protocol tips

  • Use stock solutions within 1 day
  • Optimal dye concentrations are likely to vary depending on cell type; use the highest dye concentration that gives minimal background
  • The stains in this kit do not survive fixation or permeabilization
Kangaroo rat (PtK2) cells stained with the LIVE/DEAD® Viability/Cytotoxicity Kit.