Karyotype analysis of iPSC lines

Growing iPSC cultures at about 70-80% confluence was treated with sterile colcemid (0.1 μg/mL; Sigma-Aldrich) for 2-3 h to induce metaphase arrest. The cells were then harvested using 0.25% trypsin to prepare single-cell suspensions and further treated with a hypotonic solution (0.075 M KCl), fixed using ice cold methanol and glacial acetic acid (3:1) and then dropped onto clean glass slides (Thermo Fisher Scientific Inc.) and air dried. After a brief trypsin treatment, the chromosomes were G-banded by Giemsa staining. Clear metaphase spreads (n=20) were imaged and analyzed using the CytoVision automated image analysis system (Applied Imaging).