Karyomax colcemid

Cells were seeded out into 6 cm dishes and treated with PDS for 24 h. Karyomax colcemid (50 μl of 10 μg/ml, Gibco) was added to the cells for 1–4 h to arrest cells at the metaphase stage of mitosis. Cells were then trypsinised and collected. Pre-warmed hypotonic solution (75 mM KCl) was then added to the cells and incubated for 15 min at 37°C. Cells were spun down and fixative (3:1 methanol:glacial acetic acid) was gently added dropwise to the pellet to a final volume of 10 ml. Cells were incubated at –20°C for 30 min, spun down, and fixative was replaced twice. Cells were then resuspended in an appropriate volume of fresh fixative and cells were dropped onto warmed glass slides. Spreads were hardened for 5 days at room temperature and then Telo-FISH was carried out as above. Fragile telomeres were scored when multiple telomeric signals were seen that were spatially separated from chromatid ends, as in (8 (link)).

Metaphase chromosomes were prepared as described elsewhere [14 (link)]. Cells were treated with KaryoMAX colcemid (Gibco; Cat# 15212012) at a final concentration of 75 ng/mL for 20 min to arrest cells in metaphase. Cells then were washed with PBS without Ca²⁺ and Mg²⁺ (Gibco; Cat# 14190250), followed by trypsin treatment to dislodge adherent cells, and centrifuged at 1000 rpm at room temperature. The supernatant was removed, and the cell pellet was gently resuspended in prewarmed hypotonic solution (75 mM KCl; Gibco; Cat# 10575090). Cells then were incubated in a 37°C water bath for 15 min. After hypotonic treatment, cells were fixed by gently adding 500 μL of acetomethanol fixative (3:1, methanol: acetic acid) with gentle and thorough agitation. After centrifugation for 10 min at 1000 rpm, the cell pellet was gently resuspended in 2 mL of supernatant and fixative was added in a dropwise manner with constant agitation for a total of 4 mL. An additional 4 mL of fixative was added and the cells were allowed to rest at room temperature for 20 min. After two additional washes with fixative solution, cells were dropped onto pre-cleaned glass slides at 20–25 °C with 42–45% humidity to obtain optimum spreading of metaphase chromosomes.

Fosmid clones for detection of Adam10 (WI1-600D17) were selected using UCSC Genome Browser online tool (University of California, Santa Cruz, USA), and obtained from BACPAC Resources (Children's Hospital Research Institute, Oakland, USA). mCCD_cl1 cells were cultured until confluency, treated for 10 min at 37°C with colcemid (KaryoMAX Colcemid, Gibco), trypsinized and burst (10 min at 37°C) using a hypotonic solution (KCl and sodium citrate). The cell nuclei were then fixed using a 3:1 methanol/acetic acid solution and dropped on microscope slides. FISH analysis was conducted as previously described.^{22 (link)}