HCS CellMask™ Stains Protocol
Nuclear stain for HCA/HCS cell demarcation
Useful as cell delineation reagents for HCS platforms, HCS CellMask™ Stains label the entire cell (i.e., cytoplasm and the nucleus) to provide a description of a cell’s anatomy, and provide an accurate backdrop against which the features of interest can be assessed. HCS CellMask™ stains are available in a range of fluorescent colors, they can be applied to cells immediately after fixation or in the last step of multiplexing protocols, and they are compatible with detergent-based permeabilization. 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 and cytosolic demarcation in high-content analysis/screening (HCA/HCS)
This protocol should not be used for:
- Flow cytometry
|1. Add 25 μL DMSO (Component B) to the entire contents of the HCS CellMask™ Stain (Component A) to make a 10 mg/ mL stock solution.|
|2. On the day of the assay, prepare a fixative solution by adding 2.5 mL 16% aqueous paraformaldehyde (PFA) to 7.5 mL PBS, to obtain a 4% PFA solution.|
|3. On the day of the assay, prepare a permeabilization solution by adding 10 μL Triton® X-100 to 10 mL PBS.|
|4. On the day of the assay, prepare a staining solution by adding 2 μL of the HCS CellMask™ Stain stock solution to 10 mL PBS.|
|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, and incubate as desired.|
|3. Remove the medium.|
|4. Add 100 μL fixative solution (4% PFA) to each well.|
|5. Incubate for 15 minutes.|
|6. Remove the fixative.|
|7. Wash the cells 2–3 times in PBS.|
|8. Add 100 μL permeabilization solution (Triton® X-100) to each well.|
|9. Incubate for 15 minutes.|
|10. Remove the permeabilization solution.|
|11. Wash the cells 2–3 times in PBS.|
|12. Optional: Perform antibody labeling.|
|13. Add 100 μL staining solution (2 µg/mL HCS CellMask™ Stain) to each well.|
|14. Incubate for 30 minutes, protected from light.|
|15. Wash the cells 2–3 times in PBS.|
|16. Image the cells.|
- Warm to room temperature and briefly centrifuge the DMSO solution to the bottom of the vial each time before use.
- Apply CellMask™ stains to cells immediately after fixation and permeabilization or after antibody labeling.
- Treat all nucleic acid binding dyes as potential mutagens and handled with care.
- Handle DMSO with care.
Cells stained with HCS CellMask™ Blue Stain. Alexa Fluor® 488 Click-iT® Plus EdU, and Alexa Fluor® 647 goat anti–mouse IgG and imaged with the Thermo Scientific™ CellInsight™ High-Content System.
|HCS CellMask™ Blue||HCS CellMask™ Green||HCS CellMask™ Orange||HCS CellMask™ Red||HCS CellMask™ Deep Red|
|Standard filter set||DAPI||FITC||Cy®3||Cy®3.5||Cy®5|
|EVOS® Light Cube||DAPI||GFP||RFP||Texas Red®||Cy®5|