12 well suspension plates

The capacity of BMP-4 to quantitatively promote primitive streak induction in human embryonic stem cells (hESC) was determined by using a MIXL1-GFP reporter cell line [14 (link)], as described previously [15 (link)]. In brief, single-cell suspensions were seeded in 12-well suspension plates (Greiner, Austria) at 0.33 × 10⁵ cells/ml in mTeSR medium (Stem Cell Technologies, Canada) supplemented with 10 μM Y-27632 inhibitor (kindly provided by Gerald Dräger, Leibniz University Hannover, Germany). Suspension aggregates formed within 24 h h. After 4 days, primitive streak induction was initiated by changing the medium to RPMI medium supplemented with 2% B27 supplement (Life Technologies, USA), 3 μM CHIR99021 inhibitor (kindly provided by Gerald Dräger, Leibniz University Hannover, Germany) and BMP-4 concentrations as indicated in the results section. Commercial rhBMP-4 (R&D Systems, USA) from NS0 mammalian cell culture was used as a positive control. Aggregates were harvested after 24 h and dissociated using Accutase for 5 min at 37 °C. The GFP signal was measured with a BD Accuri C6 flow cytometer (BD Biosciences, USA) and analyzed using FlowJo (v10; TreeStar, FlowJo LLC, USA).

Equilibrated PTC5000 beads were plated onto 12 well suspension plates (Greiner Bio) at 4000 beads/well in 2 mL of complete growth media. To each well 1 mL of cells were added at a concentration of 1.2×10⁵ cells/mL (mES 46C Sox-1 GFP) or 3.6×10⁵ cells/mL (hES Shef6) and left to attach for either 24 (mES) or 48 (hES) hours. For each protocol validated, two independent wells were prepared.
After the attachment period, media were decanted; beads washed twice in DMEM (in situ) and appropriate media for each stage of differentiation added. After 1 week the washing process was repeated and the media for the next stage of differentiation added. In between stages, media were refreshed by exchanging half the volume with fresh media. At the end of the differentiation period beads were washed, fixed, immunostained and analysed using COPAS (Union Biometrica Inc.)