HCS LIVE/DEAD Green Kit using Hoechst 33342 Protocol

1. Culture cells in appropriate medium in a 96-well plate

2. Add 2.1 μL Image-iT DEAD Green viability stain (Component A) to 6 mL complete medium to create staining solution

3. Add 3 mL 16% paraformaldehyde and 18 μL Hoechst 33342 (Component B) to 9 mL PBS to create fixation solution

4. Add test compound or drug to cells to a total volume of 125 μL and incubate as desired

5. Add 50 μL Image-iT DEAD Green staining solution to each well for a total volume of 175 µL

6. Incubate at 37°C for 30 minutes

7. Remove medium

11. Wash wells once with PBS

6. Incubate at 37°C for 30 minutes

7. Remove medium

8. Add 100 μL fixation solution to each well

9. Incubate at room temperature for 15 minutes

10. Remove the fixation solution

11. Wash wells once with PBS

12. Add 100 μL PBS to each well

13. Analyze cells on a high-content imaging instrument