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.

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.

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.