At the indicated time, culture was fixed and immunostained for iPSC markers Nanog and SSEA1. Stained samples were kept in phosphate buffered saline (PBS) and loaded onto ImageXpress Micro High-Content Analysis System (Molecular Devices) for automated whole well imaging at 4X magnification. The images were stitched using the MetaXpress Analysis Software (Molecular Devices) and loaded in Photoshop for colony counting. A transparent layer was created and positive colonies were marked and covered with a circular brush. The layer was then saved as a separate image and the number of circular marks were counted using ImageJ. Reprogramming efficiency was obtained by dividing the number of Nanog+ positive colonies by the total number of cells seeded at the beginning of the experiments.
For flow cytometry analysis, the culture was first treated with 500U/ml collagenase II dissolved in plain Knockout™ DMEM for 15 minutes to partially digest the ECM, followed by trypsin treatment. The detached cells was triturated with progressively smaller needles to obtain a single cell suspension before incubation with StainAlive™ SSEA1 antibody (Stemgent) for 30 minutes. The suspension was spun down and washed with PBS two times before being analyzed with Guava (Merck Millipore).
For flow cytometry analysis, the culture was first treated with 500U/ml collagenase II dissolved in plain Knockout™ DMEM for 15 minutes to partially digest the ECM, followed by trypsin treatment. The detached cells was triturated with progressively smaller needles to obtain a single cell suspension before incubation with StainAlive™ SSEA1 antibody (Stemgent) for 30 minutes. The suspension was spun down and washed with PBS two times before being analyzed with Guava (Merck Millipore).