Two-color nuclear staining assay for cell viability

This kit includes Image-iT® DEAD Green™ viability stain for discrimination of dead cells and HCS NuclearMask™ Deep Red stain or Hoechst 33342 for total cell demarcation. Once the stains in this kit are used on your live-cell sample, the signal from the stains will be retained if the sample is fixed and permeabilized.

This protocol can be used for:

  • Identifying dead cells using high-content imaging

This protocol should not be used for:

  • Flow cytometry

You will need the following for this protocol:


1. Culture cells in appropriate medium in a 96-well plate
2. Add 2.1 μL Image-iT® DEAD Green™ viability stain (Component A) to 6 mL complete medium to create staining solution
3. Add 3 mL 16% paraformaldehyde and 18 μL Hoechst 33342 (Component B) to 9 mL PBS to create fixation solution
4. Add test compound or drug to cells to a total volume of 125 μL and incubate as desired
5. Add 50 μL Image-iT® DEAD Green™ staining solution to each well for a total volume of 175 µL
6. Incubate at 37°C for 30 minutes
7. Remove medium
8. Add 100 μL fixation solution to each well
9. Incubate at room temperature for 15 minutes
10. Remove the fixation solution
11. Wash wells once with PBS
12. Add 100 μL PBS to each well
13. Analyze cells on a high-content imaging instrument

Spectral information and storage

  Image-iT® DEAD Green™ stain Hoechst 33342
Excitation/Emission 488/515 nm 350/461 nm
Standard filter set FITC or GFP  
Storage conditions –20°C –20°C


Protocol tips

  • This stains in this kit are compatible with antibody labeling protocols
  • This kit provides sufficient material for two 96-well plates

Graph showing valinomycin dose-response as detected using HCS LIVE/DEAD Green Kit reagents
Dose-response for valinomycin in HeLa cells using the HCS LIVE/DEAD® Green Kit.