1. Culture cells in appropriate medium in a 96-well plate
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:
- Cells growing in flat-bottom 96-well plates
- HCS LIVE/DEAD Green Kit (Cat. No. H10290)
- Phosphate-buffered saline (PBS) (Cat. No. 14190-144)
- Paraformaldehyde, 16% aqueous solution
- High-content imaging instrument with FITC and Cy5 filters
Protocol
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 | Cy5 |
| EVOS Light Cube | GFP | Cy5 |
| Storage conditions | –20°C | –20°C |