Metaphase spreads and spectral karyotyping of human embryonic stem cells

INTRODUCTIONSpectral karyotyping is a powerful technique for enumeration of chromosomes in plant, animal, and human cells with high precision. It is also used as a tool for detecting and diagnosing abnormalities in individual chromosomes, such as deletions, duplications, and translocations. There are three basic steps to obtaining good spectral karyotypes: (1) making metaphase spreads, (2) hybridizing probes to the DNA, and (3) analyzing the spectral characteristics of the chromosomes. There are two techniques for spectral karyotyping that are widely used: spectral karyotype (SKY) and multicolor fluorescence in situ hybridization (M-FISH). Both techniques use multicolored fluorescent probes which emit at different wavelengths (200-700 nm) upon light excitation to hybridize to the target DNA sequences. The methods differ in the way in which light is captured and analyzed by the corresponding software. In this protocol, methods for making metaphase spreads from human embryonic stem cells (hESCs) for spectral karyotyping (SKY) using the spectral imaging system and software from Applied Spectral Imaging are described.