Fig 2. Outline of human quantitative embryoid body (hqEB) system strategies.

(A) Preparation of spheroids from TRA1-60+ magnetically purified hESCs. Low passage H9 cells were dissociated to single cells by Accutase treatment, then TRA1-60+ cells were magnetically purified. These were then plated at known density into Aggrewell plates to from spheroids of uniform size. (B) Alternative plating and differentiation approaches. Three methods were assessed: (1) Spheroids were deposited individually into cell culture plate wells, and imaged every 3 days. (2). Pools of EBs are plated into 6 well dishes under non-adherent conditions, imaged, quantitated for size. (3) Spheroids are attached onto Matrigel coated plates and allowed to proliferate and spread under adherent conditions.