5-To test this idea, the authors set up a five-stage route for human embryonic stem cells. The cells moved from definitive endoderm to gut like and pancreatic progenitor stages, then on to endocrine cells that make pancreatic hormones. Early on, the team used real-time PCR to follow shifts in more than thirty lineage markers and see which cells stayed on a pancreatic path. They then checked protein patterns with immunofluorescence and western blots, with flow cytometry and ELISA as broader readouts. Finally, they measured insulin content and C-peptide release and examined secretory granules by electron microscopy to judge how β-cells-like these cells had become.