Anti tra1 60 vio488

HiPSCs and hiPSC-CM were dissociated as single cells using TrypLE™ Select Enzyme (1x) (Gibco) and TrypLE™ Select Enzyme (10x) (Gibco), respectively. For hiPSCs 2 × 10⁵ cells were fluorescently labelled for pluripotent stem cell surface markers using 1:20 anti-SSEA4-VioBlue (Miltenyi Biotec, Bergisch Gladbach, Germany) 130-098-366) and 1:600 anti-Tra1-60-Vio488 (Miltenyi Biotec, 130-106-872) antibodies. Then, cells were fixed and permeabilized using the FoxP3 Staining Buffer Set (Miltenyi Biotec, 130-093-142) and labelled for nuclear markers using 1:50 anti-Oct3/4-APC (Miltenyi Biotec, 130-123-318) and 1:100 anti-Nanog-PE (Cell Signaling, Danvers, MA, USA, 14955S). For the characterization of cardiac markers 2 × 10⁵, hiPSC-CMs were fixed and permeabilized similarly to the hiPSCs and fluorescently labelled using 1:50 anti-cardiac Troponin T-FITC (Miltenyi Biotec, 130-119-575) and 1:10 anti-MLC2v-APC (Miltenyi Biotec, 130-106-134) antibodies. Isotype stainings were performed as controls. Cells were measured for marker expression using a MACSQuant VYB (Miltenyi Biotec) flow cytometer and data analyzed using FlowJo software.

For staining, iPSCs were grown in 96-well imaging plates (CellCarrier, PerkinElmer) in E8 medium. After 3 days, cells were fixed with Cytofix reagent, followed by blocking and permeabilization with PermWash reagent (both from BD Biosciences). Then, cells were incubated with the dye-conjugated antibodies anti–TRA-1-60–Vio488 (Miltenyi Biotec, REA157, 130-106-872), anti-SSEA4–PerCP-Vio700 (Miltenyi Biotec, REA101, 130-105-053), anti-OCT3/4 (Isof. A)–APC (Miltenyi Biotec, REA338, 130-105-555), and anti-NANOG (D73G4)–PE (Cell Signaling Technology, 14955). Nuclei were stained with Hoechst 33342 (2.5 μg/ml in PBS; Invitrogen). Images were captured using an Operetta high-content imaging system (PerkinElmer).