SKY analysis of ES cells was performed. Briefly, metaphase chromosome were prepared from exponentially growing cells after treatment with colcemid (KaryoMAX, GIBCO) at 0.1 μg/ml for 1 hr53 (link). Cells were swollen in prewarmed 0.56% KCl for 10 min at 37°C, then carefully fixed in methanol:acetic acid (3:1) overnight and kept at -20°C. Metaphase spreads were prepared by dropping cells in the fixative onto chilled Superfrost glass slides (Fisher Scientific) at 25°C and 60% of humidity. After air drying and pepsin digestion, slides were denatured at 80°C for 5 min, hybridization was performed using SKY probe (Applied Spectral Imaging, San Diego) and fluorescence-conjugated secondary antibodies in accordance with the manufacturer’s specification. Metaphase images were captured and analyzed using a SpectraCube® imaging system and software (Applied Spectral Imaging). At least 20 metaphases from each cell line were scored for chromosomal aberration.
Spectracube imaging system and software
Manufactured by Applied Spectral Imaging
The SpectraCube® is an imaging system and software solution developed by Applied Spectral Imaging. The system captures and analyzes spectral data, providing detailed information about the composition and characteristics of the imaged samples. The SpectraCube® system and software enable users to collect and process spectral data for various applications.
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2 protocols using spectracube imaging system and software
1
Karyotyping of Embryonic Stem Cells
2
Karyotyping Embryonic Stem Cells via SKY
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SKY analysis of embryonic stem cells was performed. Briefly, metaphase chromosome were prepared from exponentially growing cells after treatment with colcemid (KaryoMAX, GIBCO) at 0.1 μg ml−1 for 1 hr (ref. 51 (link)). Cells were swollen in prewarmed 0.56% KCl for 10 min at 37 °C, then carefully fixed in methanol:acetic acid (3:1) overnight and kept at −20 °C. Metaphase spreads were prepared by dropping cells in the fixative onto chilled Superfrost glass slides (Fisher Scientific) at 25 °C and 60% of humidity. After air drying and pepsin digestion, slides were denatured at 80 °C for 5 min, hybridization was performed using SKY probe (Applied Spectral Imaging, San Diego) and fluorescence-conjugated secondary antibodies in accordance with the manufacturer's specification. Metaphase images were captured and analysed using a SpectraCube imaging system and software (Applied Spectral Imaging). At least 20 metaphases from each cell line were scored for chromosomal aberration.
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