Table 3.

Troubleshooting table

Step	Problem	Possible Causes	Solutions
5	Colonies not coming off	Pipetting too gently	Use a strong motion to rinse most cells off in one shot.
		Insufficient treatment	Extend treatment time
	Too many colonies come off during EDTA treatment	Treating for too long	Shorten treatment time or skip one washing step.
		Plate is disturbed too much during buffer change	Don’t shake or swirl the plate during the treatment.
			Cells can be neutralized and collected with E8 medium. Either spin down and plate, or directly plate the suspension and change medium again after 1 h
8	Poor cell survival	Cells have not been passaged frequently enough	Cells need to be passaged around every 4 d. If the culture is beyond 5 d, add ROCK inhibitor
		Cells are overconfluent	Passage the cells when the cells reach to 80% confluence. ROCK inhibitors could be added to help the survival.
		Too much pipetting	Don’t repetitively pipet the cells. After sufficient treatment, cells are easily washed off the plate.
		Overtreatment leads to too many single cells	Shorten the treatment time or skip one washing step.
		EDTA concentration is too high	Decrease the EDTA concentration from 5 to 0.5–2 mM.
9	Poor pluripotency marker expression	Reagent quality	Batch test for reagents listed in Table 2.
17	Putative iPSCs do not come off the plate	Confluent plates need more EDTA to remove calcium	Increase EDTA treatment time or add more washing steps.
			If iPSC colonies are rare, aim at potential iPSC colonies to specifically remove iPSCs.
	Poor cell survival	Cells are overconfluent	Add ROCK inhibitor.
26	No attached colony	Colony is too big	Cut the colony to small pieces before harvest.
		Non-iPSC morphology	Colonies picked may not have been iPSC colonies, improve iPSC identification
27	Poor survival	Cells are overconfluent	Add ROCK inhibitor
			Move the culture to hypoxia condition (5% O2)