HCS Live/Dead Green Kit

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-red

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

Make

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

Add-red

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

Clock-30

5. Incubate at 37°C for 30 minutes

Remove

6. Remove medium

Add-gray

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

Clock-15

8. Incubate at room temperature for 15 minutes

Remove-gray

9. Remove the fixation solution

Wash

10. Wash wells once with PBS

Add-gray

11. Add 100 μL PBS to each well

Image

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_

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

Spectral information and storage

 Image-iT DEAD GreenHCS NuclearMask Deep Red
Excitation/Emission488/515 nm638/686 nm
Standard filter setFITC or GFPCy5
EVOS Light CubeGFPCy5
Storage conditions–20°C–20°C