HCS NuclearMask Deep Red Stain Protocol

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.

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.

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