Table 2.

Troubleshooting table

Step	Problem	Possible reason	Solution
7	Poor cell survival after thawing	Cell thawing process was too slow and/or cells were not plated in media supplemented with CEPT	Ensure cells are thawed within 5 min and plate in media supplemented with CEPT for maximum cell survival
17	Poor cell survival after passaging	Passaging induces several cell stress mechanisms that may reduce cell survival upon passaging	Use CEPT consistently at each passage to reduce cell stress and increase quality of cell line
22	Cells are not Calcein AM positive after 2h	Calcein AM concentration may be too low	Increase Calcein AM concentration to 1:25,000 (40 nM) and repeat steps 19–21
33	Cell suspension has aggregates or clumps after straining	Cell culture was overgrown or too confluent (> 80%) at the time of dissociation.	Only use cell cultures that are ≤ 80% confluent
			Remove cell clumps by centrifugation at 50 g for 30 seconds. Without disturbing cell pellet, take cell suspension supernatant from top. Supernatant will have fewer aggregates or clumps.
41	Dispensed droplets have multiple cells	Cell suspension is too dense (> 5,000 cells/mL) or flow rate is too high (≥ 2 cells/s)	Dilute cells suspension until flow rate is ≤ 2 cells/s
47	Low single-cell cloning efficiency	Cells were not adapted to StemFlex media prior to single-cell dissociation	Ensure cell culture is adapted to StemFlex media for at least 24 h prior to single-cell dissociation
		Cells were aspirated during media changes	Aspirate media from side of well and avoid contact with the bottom of the well
		CEPT was not added to StemFlex media	Ensure StemFlex single-cell plating media is supplemented with CEPT
47	No colonies	High temperatures generated during imaging killed cells	Reduce imaging intervals
			Use alternative imaging platforms that do not generate too much heat during the imaging process