Image of HCS NuclearMask™ Stain packaging

Nuclear stain for HCA/HCS cell demarcation

The HCS CellMask 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 Red Stain (Cat. No. H10326)
  • Flat-bottom 96-well microplates
  • Optional: Paraformaldehyde, 16% aqueous solution
  • Phosphate-buffered saline (PBS)
  • HCA instrument or fluorescence microscope

Protocol

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 10 μL HCS NuclearMask 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 10 μL HCS NuclearMask 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.
Spectral information and storage
 HCS NuclearMask Red
Excitation/Emission (nm)622/645
Standard filter setTexas Red
EVOS Light CubeTexas Red
Storage conditions≤–20°C

 

 

Protocol tips

  • 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 1,000X concentrate in DMSO.

cells stained with HCS NuclearMask™ Red Stain
Cells stained with HCS NuclearMask Red Stain and imaged with the Thermo Scientific CellInsight High-Content System.