Chromosome Karyotyping Protocol

The metaphases were arrested by incubation with Colcemid (#15210-040, KaryoMax Colcemid Solution (Invitrogen, Carlsbad, CA, USA) (10 ug/mL) 2 h prior to harvest. Cells were collected and treated with hypotonic solution (#685804, KCL 0.075M, Macron Chemical, Radnor, PA, USA) for 15 min at 37 °C and fixed with 3:1 ratio of methanol: acetic acid. Slides were prepared and aged overnight for use in G-band, SKY analysis. Chromosomes were stained with a trypsin- Giemsa staining technique [22 (link)]. Analyses were performed under an Axioplan ImagerZ2 (Zeiss, Dublin, CA, USA) microscope coupled with a CCD camera; images were captured with ASI software, (Applied Spectral Imaging Inc., Vista, CA, USA). The karyotype was determined by comparison to the standard ideogram of banding patterns for human chromosomes by ISCN 2009.