Proteome profiler human pluripotent stem cell array kit

Stem cell-specific marker proteins were measured using the Proteome Profiler Human Pluripotent Stem Cell Array Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. In brief, the hiPSCs were seeded on a feeder layer of MEF cells (ReproCELL Inc., Kanagawa, Japan) and seeded at 1.5 × 10⁵ cells per 10-cm plate. iPSCs were cultured in 100 mm dishes in Primate ES Cell Medium (ReproCELL Inc., Kanagawa, Japan) to ∼80% confluence. To prepare sampling hiPSCs for cell assay, the hiPSCs were first detached from the feeder layer and partially dissociated as described for maintenance passage. Next, the contaminating MEF cells were removed by incubating the cell suspension on a gelatin-coated plate at 37°C for 2 h in Primate ES Cell Medium (ReproCELL Inc., Kanagawa, Japan) with 10 μM Y-27632(257–00511: FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) to ensure the high purity of the hiPSCs. 1 mL cell lysate from the 100 mm dish was prepared using an EzRIPA Lysis kit according to the manufacturer’s instructions (ATTO, Tokyo, Japan).