Metaphase Chromosome Preparation and Immunostaining

Metaphase chromosomes were prepared essentially as described previously (Chadwick and Willard 2002 (link)). Briefly, a 1:400 dilution of KaryoMAX Colcemid (Thermo Fisher Scientific, Cat. No. 15212-012) was added to media of actively dividing cells and incubated for one hour at 37°C 5% CO₂. The media was collected and transferred to a 50ml tube. Cells were washed with phosphate buffered saline (PBS) which was added to the media in the 50ml tube. Cells were detached with TrypLE (Thermo Fisher Scientific, Cat. No. 12605-010) and collected using the media/PBS mixture, before counting followed by briefly pelleting the cells. Chromosomes were attached to slides by centrifugation using a Shandon Cytospin 4 (Thermo Fisher Scientific), spinning at 1,900 × rpm for 10 minutes. Post-spin, samples were fixed for 10 minutes in 1x PBS supplemented with 0.1% (v/v) Triton X-100 and 3.7% formaldehyde, before washing twice in 1x PBS. Samples were then subjected to immunofluorescence as described below.