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Table 5 Troubleshooting

From: A protocol to assess cell cycle and apoptosis in human and mouse pluripotent cells

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.