Table 5.

Troubleshooting

Step	Problem	Possible reason and solution
	Low number of cells in S Phase	EdU substrate incubation too short or batch of substrate used too many times. Use fresh aliquoted EdU and incubate 15-30 minutes longer.
	No distinct G1/S/G2M phases	Cells not healthy or DAPI staining not working. Check that cells look healthy before starting experiment and leave DAPI staining overnight at 4°C (in the fridge). Also consider using a modified method using Hamelik and Krishan protocol by lysing cells (Hypotonic solution) for 1 minute followed by centrifugation and then using EdU reaction (Click iT)[20].
	DilC % apoptosis lower than expected or not working.	Check with CCCP control there is a shift in DilC positive cells to negative. Keep cells at 37°C at all times throughout the procedure and analyse immediately following PI staining.