Day (−1): iPSCs were washed with room temperature 1X DPBS (minus Ca2+ and Mg2+) once. Wash was aspirated to waste and TrypLE Select (1X; 37°C; 1 ml per 6-well) and placed in incubator (5% CO2, 37°C). After 3–5 minutes, cell plate was placed in cell culture hood and the side of the plate was lightly tapped to dislodge loosely adherent iPSCs for 30 seconds. After lightly tapping the plate, 1 ml of room temperature 1X DPBS (minus Ca2+ and Mg2+) was added to each 6 well plate. Cells were collected in to a 15-ml conical tube (Corning) using a 10-ml Stripette® (Corning). Cells were centrifuged at 200 × g for 5 minutes at room temperature. After centrifugation, supernatant was aspirated to waste and cells were suspended in E8 medium + Y-27632 ROCK Inhibitor (RI,10 μM; R&D Systems), and gently triturated to generate a single-cell suspension, counted, and cell density adjusted to seed at 5 × 105 cells/cm2 in tissue-culture treated 6-well plates. Final volume was adjusted to 1.5 ml of E8+ RI. Cells were cultured for 24 hours under normoxic conditions (20% O2, 37°C).
