Hyper spectral imaging system

To evaluate chromosome instability (CIN), SKY analysis was performed as described previously (31 (link)). Briefly, organoids were disassociated from Matrigel by incubating with Dispase II, and single-cell suspensions were generated by incubating with TrypLE (ThermoFisher Scientific). Cells were then arrested in metaphase by incubating with Colcemid™ (10 µg/mL; 15210-040, KaryoMAX ® Colcemid Solution, Invitrogen, Carlsbad, CA) for 3 hours, treated with hypotonic solution (KCl 0.075M, 6858-04, Macron Chemical) for 15 minutes at 37°C, and then fixed with methanol:acetic acid 3:1. The prepared metaphase spread slides were aged overnight and then hybridized with the 21-color mouse SKY paint kit (FPRPR0030, ASI) in a humidity chamber at 37°C for 16 hours (34 (link)). Spectral images were acquired using a Hyper Spectral Imaging System (Applied Spectral Imaging Inc., CA) mounted on top of an epi-fluorescence microscope (Imager Z2, Zeiss) and analyzed using HiSKY 7.2 acquisition software (GenASIs, Applied Spectral Imaging Inc., CA). An average of 10–15 mitoses of comparable staining intensity and quality were examined per organoid line and evaluated for chromosomal abnormalities.

Treatment with Colcemid (10 μg/ml, KaryoMax, Invitrogen, Carlsbad, CA, USA), hypotonic treatment (0.075 M KCl), fixation with methanol/acetic acid (3,1), slide preparation and G-banding were performed as previously described [20 ] and the images were captured and analyzed with the HiBand system (Applied Spectral Imaging, Carlsbad, CA, USA). For spectral karyotyping (SKY), the slides were processed using the 24-color Human SKY Paint kit (Applied Spectral Imaging) according to the manufacturer’s protocol. Spectral images of the hybridized metaphases were acquired using the HyperSpectral Imaging system (Applied Spectral Imaging) and analyzed using the HiSKY v.7.2 acquisition software (Applied Spectral Imaging). All cell lines were analyzed by G-band karyotyping. hTERT-CRL-2097, hTERT-CRL-2097 + EML4-ALK, its derived soft-agar clones #1 and #2, and HBET1 were also analyzed by SKY.

XSCID CD34⁺ cells gene edited were prepared for chromosomal analysis and FISH as previously described. The metaphases were hybridized with the 24-color human SKY paint kit (FPRPR0028, ASI). Twelve metaphases were imaged for karyotype. Spectral images of the hybridized metaphases were acquired using Hyper Spectral Imaging system (Applied Spectral Imaging) mounted on top of an epi-fluorescence microscope (Imager Z2, Zeiss). Images were analyzed using HiSKY 8.2 acquisition software (Genasis, Applied Spectral Imaging). G-banding was simulated by electronic inversion of DAPI counterstaining. An average of 10–15 mitoses of comparable staining intensity and quality was examined per cell line and compared for chromosomal abnormality. The karyotype was determined by comparison to the standard ideogram of banding patterns for human chromosomes.