Accutase

Protein induced pluripotent stem cells (piPS, System Biosciences, USA), were cultured on Gelatin (Sigma-Aldrich, St. Louis, MO, USA) coated dishes with feeder layer of mitomycin C (Sigma-Aldrich, USA) inactivated mouse embryonic fibroblasts (MEFs) in serum-free iPS medium DMEM/F12 supplemented with 20% KSR, 2 mM GLUTAMAX, 100 µM non-essential amino acids, 100 U/mL/100 µg/mL Penicillin/Streptomycin, 10 ng/mL bFGF (all from ThermoFisher Scientific, USA) and 100 µM -mercaptoethanol (Sigma-Aldrich). The medium was changed every day. When the culture reached about 80% confluence, iPS cells were subcultured with Accutase (Lonza, Basel, Switzerland) in proportion 1:4–1:10 and seeded onto fresh feeder layer in presence of 10 µM ROCK inhibitor Y-27632 (Sigma-Aldrich). For immunocytochemical stainings, iPS cells were cultured in feeder-free conditions on growth factor-reduced Matrigel (Corning, New York, NY, USA) coated dishes in MEF-conditioned iPS medium. MEFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; 4.5 g/L glucose; Lonza), supplemented with 10% v/v FBS, 2 mM L-glutamine and 100 U/mL/100 µg/mL Penicillin/Streptomycin antibiotics solution (all from ThermoFisher Scientific, Waltham, MA, USA).

H9 cells from Vitronectin plates were dissociated to single cells using Accutase (ThermoFisher Scientific) after being incubated with 10 μM ROCK inhibitor (Y27632) for at least 1 h. After diluting the Accutase with E8 media, the cells were counted and 200,000 cells per reaction were spun down (300 xG, 5 min) and resuspended in premixed Nucleofection solution, P3 (Lonza). Twenty micro-liters of cell suspension was added to a maximum of 2 μg of total DNA (no more than 2 μL) and transferred to a Nucleofection chamber (16-well strip). The H9 cells were transfected using parameter CM 130, P3 solution, and plated out on Vitronectin coated plates (35 mm, Nunc) in E8 media supplemented with 10 μM ROCK inhibitor. Media was changed to regular E8 media after 1–2 days.