Amniomax medium

Primary fibroblasts were seeded into AmnioMAX medium (Life Technologies) to achieve ∼60% confluency after 24 h. Cells were then incubation with 10 μM BrdU for 30 min, washed, harvested, and fixed with 70% EtOH for 16 h at −20°C. Fixed cells were then digested with 1 mg/mL pepsin, denatured in 2 M HCl for 15 min, and washed with PBS. After blocking in 0.5% BSA and 0.5% Tween-20, BrdU labeling was detected using anti-BrdU antibody (1:75; Abcam ab6326) and anti-rat Alexa fluor 488 secondary antibody (Thermo Fisher Scientific A11006). DNA content was determined by costaining with 50 μg/mL propidium iodide. Cells were assayed on a BD Biosciences LSR Fortessa flow cytometer and data were analyzed using FlowJo software (v7.6.1, Tree Star).

The tail tips were cut from each animal and used for cell culture. Metaphase chromosome spreads were prepared from fibroblasts of tail tissue following the protocol described in Ezaz et al. [23] (link). Briefly, minced tail tissues were implanted in a T25 culture flask containing AmnioMax medium (Life Technologies, Carlsbad, California, USA) and were allowed to propagate under the condition of at 28°C and in an atmosphere of 5% CO₂. Once the fibroblasts had grown to about 80% confluency, cultures were split intoT75 flasks and subsequently split up to four passages before the chromosomes were harvested. Colcemid (Roche, Basel, Switzerland) was added to the culture flask to a final concentration of 75 ng/ml prior to harvesting. Cultured cells were harvested by trypsin treatment, suspended in 0.075 M KCl, then fixed in 3∶1 methanol:acetic acid and the cell suspension dropped onto glass slides and air-dried.