6. Add 200 ml of fixing solution to all wells, wrap the plate in aluminum foil and fix the cells overnight at +4 °C.
7. Discard fixing solution and wash the cells three times with sterile PBS, 200 ml/well.
8. Reconstitute Nuclease reagent with 26 ml of sterile water. To this, add 260 ml of anti-BrdU monoclonal antibody. Add 50 ml to all wells.
9. Incubate plate for 45 min at 37 °C in 5% CO2.
10. Discard Nuclease reagent and wash the cells three times with sterile PBS, 200 ml/well.
11. Reconstitute the Cy5 labeled goat anti-mouse reagent with 275 ml of sterile water.
12. Dilute the reconstituted Cy5 labeled goat anti-mouse reagent 1:100 with sterile PBS. Add 50 ml to all wells.
13. Incubate plate for 1 h at room temperature in the dark (or cover the plate in aluminum foil).
14. Discard Cy5 labeled goat anti-mouse reagent and wash the cells three times with sterile PBS, 200 ml/well.
15. Dilute Hoechst nuclear dye to 2 mM with sterile PBS. Add 100 ml to all wells and incubate for 30 min at room temperature, in the dark.
16. Discard Hoechst nuclear dye and wash the cells twice with sterile PBS, 200 ml/well.
17. Leave the cells in 100 ml of sterile PBS. Plates can be stored at +4 °C (in the dark) for up to 1 week.
Image the fixed-cell plate on the IN Cell Analyzer 1000. Select the following filter sets for image acquisition excitation 360 and 620 nm, emission 535 and 700 nm.
Dichroic D/Cy5. Analyze the images from IN Cell Analyzer 1000 using the Object Intensity Analysis Module to quantitatively measure the nuclear fluorescence intensity per cell.
BrdU incorporation was determined as de