Image of HCS CellMask™ Stains packaging

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

You will need the following for this protocol:

  • Cells growing in culture
  • HCS CellMask™ Stains (H32712, H32713, H32714, H32720, H32721)
  • Flat-bottom 96-well microplates
  • Paraformaldehyde, 16% aqueous solution
  • Triton® X-100
  • Phosphate-buffered saline (PBS)
  • HCA instrument or fluorescence microscope


Preparing solutions

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.

Preparing solutions

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. 



Protocol tips

  • 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
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.

Spectral information and storage
  HCS CellMask™ Blue HCS CellMask™ Green HCS CellMask™ Orange HCS CellMask™ Red HCS CellMask™ Deep Red
Excitation/Emission (nm) 346/442 493/516 556/572 588/612 650/655
Standard filter set DAPI FITC Cy®3 Cy®3.5 Cy®5
EVOS® Light Cube DAPI GFP RFP Texas Red® Cy®5
Storage conditions ≤–20°C ≤–20°C ≤–20°C ≤–20°C ≤–20°C