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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:

Protocol

Grow-red

1. Culture cells in appropriate medium in a 96-well plate

Add-2

2. Add 2.1 μL Image-iT® DEAD Green™ viability stain (Component A) to 6 mL complete medium to create staining solution

Add-2

3. Add 3 mL 16% paraformaldehyde and 18 μL Hoechst 33342 (Component B) to 9 mL PBS to create fixation solution

Add-gray

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

Add-gray

5. Add 50 μL Image-iT® DEAD Green™ staining solution to each well for a total volume of 175 µL

Clock-30

6. Incubate at 37°C for 30 minutes

Remove

7. Remove medium

Add-gray

11. Wash wells once with PBS

Clock-30

6. Incubate at 37°C for 30 minutes

Remove

7. Remove medium

Remove-gray

8. Add 100 μL fixation solution to each well

Clock-15

9. Incubate at room temperature for 15 minutes

Remove

10. Remove the fixation solution

Wash

11. Wash wells once with PBS

Add-gray

12. Add 100 μL PBS to each well

Image

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  
EVOS® Light Cube GFP DAPI
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
Dose-response_

Dose-response for valinomycin in HeLa cells using the HCS LIVE/DEAD® Green Kit.