Nuclear stain for HCA/HCS cell demarcation
The HCS CellMask™ Deep Red Stain is used to measure DNA content and perform cell demarcation in both live and fixed cells on high-content imaging and analysis (HCS) platforms. The versatile HCS NuclearMask™ Stains survive standard formaldehyde-based fixation and detergent-based permeabilization methods. This protocol produces sufficient stain for one 96-well plate at a staining volume of 100 μL/well.
This protocol can be used for:
- Nuclear demarcation in high-content analysis/screening (HCA/HCS)
This protocol should not be used for:
- Flow cytometry
You will need the following for this protocol:
- Cells growing in culture
- HCS NuclearMask™ Deep Red Stain (Cat. No. H10294)
- Flat-bottom 96-well microplates
- Optional: Paraformaldehyde, 16% aqueous solution
- Phosphate-buffered saline (PBS)
- HCA instrument or fluorescence microscope
Labeling live cells
|1. Culture cells in an appropriate medium and vessel for HCA or fluorescence microscopy.|
|2. Optional: Add a test compound or drug to the cells to a total volume of 125 μL, and incubate as desired.|
|3. Prepare a staining solution by adding 40 μL HCS NuclearMask™ Deep Red Stain to 10 mL complete medium.|
|4. Remove the medium.|
|5. Add 100 μL staining solution to each well.|
|6. Incubate for 30 minutes, protected from light.|
|7. Optional: Fix the cells using paraformaldehyde. (Follow the steps below pertaining to the application of 4% PFA.)|
|8. Image the cells.
Labeling fixed cells
|1. Culture cells in an appropriate medium and vessel for fluorescence microscopy.
|2. Optional: Add a test compound or drug to the cells, and incubate as desired.
|3. Prepare a fixative solution by adding 2.5 mL 16% aqueous paraformaldehyde (PFA) to 7.5 mL PBS, to obtain 10 mL of 4% PFA.
|4. Remove the medium.|
|5. Add 100 μL fixative solution (4% PFA) to each well.
|6. Incubate for 15 minutes.|
|7. Remove the fixative.
|8. Wash the cells 3 times in PBS.|
|9. Prepare a staining solution by adding 40 μL HCS NuclearMask™ Deep Red Stain to 10 mL PBS.
|10. Remove the PBS.
|11. Add 100 μL staining solution to each well.
|12. Incubate for 30 minutes, protected from light.
|13. Image the cells.
|HCS NuclearMask™ Deep Red|
|Excitation/Emission (in nm)||638/686|
|Standard filter set||Far red|
|EVOS® Light Cube||Cy®5|
- Warm to room temperature and briefly centrifuge the DMSO solution to the bottom of the vial each time before use.
- Treat all nucleic acid binding dyes as potential mutagens and handled with care.
- This stain is supplied as a 250X concentrate in DMSO.
Cells stained with HCS NuclearMask™ Deep Red Stain and imaged with the Thermo Scientific™ CellInsight™ High-Content System.