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 test compound or drug to cells to a total volume of 125 μL and incubate as desired

3. Make staining solution by adding 2.1 μL Image-iT® DEAD Green™ viability stain (Component A) and 40 μL  HCS NuclearMask™ Deep Red stain (Component C) to 6 mL complete medium

4. Add 50 μL staining solution to each well for a total volume of 175 μL

5. Incubate at 37°C for 30 minutes

6. Remove medium

7. Add 100 μL fixation solution (16% paraformaldehyde) to each well

8. Incubate at room temperature for 15 minutes

9. Remove the fixation solution

10. Wash wells once with PBS

11. Add 100 μL PBS to each well

12. Analyze cells on a high-content imaging instrument


  Protocol tips

  • This stains in this kit are compatible with antibody labeling protocols
  • This kit provides sufficient material for two 96-well plates
Dose-response for valinomycin in HeLa cells using the HCS LIVE/DEAD® Green Kit.

Spectral information and storage

  Image-iT® DEAD Green™ HCS NuclearMask™ Deep Red
Excitation/Emission 488/515 nm 638/686 nm
Standard filter set FITC or GFP Cy®5
EVOS® Light Cube GFP Cy®5
Storage conditions –20°C –20°C