sho-probes-generic-pouch

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 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