Skyview imaging software

Metaphase chromosome spreads were prepared from untreated CAL-33 parental cell line and generated CAL-33 sublines. Colcemid (Roche) was added at a final concentration of 0.1 μg/ml to the culture medium of exponentially growing cells at a density of 6 × 10⁶ cells per 75 cm². After 3 h of incubation time, cells were washed with PBS, trypsinized, suspended in fresh culture medium followed by hypotonic KCl treatment (75 mM) at 37 °C for 25 minutes. Following centrifugation, cells were resuspended in 2–3 ml of fixation solution and approximately 40–50 μl of cell suspension was dropped on several microscope slides. After one week of ageing at room temperature, spectral karyotyping was performed as described by Hieber et al. [14 (link)]. The karyotype of each cell line was determined based on a minimum of 15 metaphases. Chromosomal aberrations were detectable by color junctions within affected chromosomes. Spectral imaging and image analysis were performed with a SpectraCube system and SkyView imaging software (both from Applied Spectral Imaging).

Cells were incubated in 200 ng/mL Colcemid (KaryoMAX Colcemid solution; Gibco) for 4 h and then incubated in 75 mM KCl. Chromosomes were subsequently fixed in 3:1 methanol:acetic acid and dropped onto glass slides. Metaphase spreads were incubated with mouse SkyPaint kit probe (Applied Spectral Imaging, Carlsbad, CA, USA) according to the manufacturer’s protocol. Image acquisition was performed using a SpectraCube system, and analyses were accomplished using the SkyView imaging software (Applied Spectral Imaging). A minimum of 20 metaphases were analyzed for each cell line.