Cx5 high content screening platform

For EB formation assays, 1.8 × 10⁵ cultured PSCs were detached from the plates using a cell scraper (Iwaki), dissociated by pipetting, transferred to low-attachment 6-well plates (Corning), and cultured with Es6 medium with 10 µM RI for 1 day and with Es6 medium alone for 13 days for EB formation. The medium was changed every 3 days. The sizes and numbers of EBs derived from PSCs on days 2 and 14 were measured using a CX5 high-content screening platform (Thermo Fisher). Gene expression levels were determined using RT-qPCR with a TaqMan Scorecard Panel (A15870), and gene expression profiles were determined using a RT-qPCR device (QuantStudio 12 K Flex).

Cells cultured on VTN-N–coated plates with 4 mL Es8 medium were washed once with PBS (−) 24 h after transfection with either siRNA or mRNA. Cells were then detached using a cell scraper (Iwaki), dissociated by pipetting, transferred into low-attachment 6-well plates (Corning, NY, USA), and cultured with Es6 medium with 10 µM ROCK inhibitor for 1 day and with Es6 medium alone for 3 days for EB formation. The sizes and numbers of EBs derived from PSCs were evaluated on day 3 using a CX5 high-content screening platform (Thermo Fisher).