Images were captured and quantified using a Cellomics Array Scan imaging system. Briefly, images were taken in 24-well plates. Individual plates were used to either measure pluripotency markers or markers to assess differentiation for one of the germ layers. Each plate contained cells from one or two cell lines, as well as technical replicates for each measurement. Three types of plate layouts were considered throughout the project: Two-channel, three-channel, and three-channel with single staining. For all layouts, the signal from the DAPI staining was read in the first channel. The first columns of each plate were used for marker staining; subsequent columns (one or two) were stained with the secondary antibody to measure background signal (Extended Data Fig. 1).
