Table 1.

Aggregate Analysis (Basic Protocol 1)

	Description	Day 1	Day 2	Day 3 and onward
Aggregates present	Small aggregates (left two aggregates in Fig. 1B)	Continue with step 22 to feed the cells	Check that aggregates are still present and have maintained their size or enlarged slightly (Fig. 1B, right aggregate) and then continue with step 29 to feed the cells	Confirm that aggregates are appropriately attached to the plate and have normal morphology and edges (Fig. 1C)
No aggregates visible	Cells appear as single cells or very small clusters of cells (4-10 cells) attached to the plate	If the medium is clear and no “sheets” of cells are visible, continue with step 22 and check the wells again at day 2 (aggregates could be too small to see)	Check to see if aggregates are now visible (Fig. 1B). If some aggregates are present, continue with step 29 to feed cells. If no aggregates are present, discard and try again, handling the cells more gently and/or plating at a higher density.
Cells are all in “sheets” or a film across the surface of the well	Cells appear as single cells floating in the medium, or medium is cloudy, with cells that are floating but stuck together (sheets) without contamination	Cells either 1) were plated too densely, and it is likely that aggregates will not form, or 2) are just in single-cell format and will not survive or form aggregates. Try continuing with step 22 and check again at day 2 for aggregates.	If small aggregates are now visible, continue with step 29 to feed the cells. If aggregates are still not visible, the cells were plated too densely, and aggregates will not form. Discard and try again, plating at a lower density.