Chromosome Analysis of Agnospheres

Chromosome analysis by G banding was performed on agnospheres. In order to increase the number of metaphases, cells were synchronized with Synchroset (Euroclone S.p.A.) according to manufacturer’s instructions and blocked with colcemid (10 μl/ml) for 1 h. Chromosome harvesting was carried out according to standard procedures. Briefly, cells were incubated in 0.075 M KCl hypotonic solution at 37 °C for 10 min, fixed in methanol–glacial acetic acid (3:1), dropped onto glass slides and dried using specific conditions for optimal chromosome spreading. G banding was performed by incubating slides in 2× SSC at 68 °C for 2 min and eventually stained with Wright’s solution for 2 min (ref. ^{57 (link)}). Metaphase images were captured using an Olympus BX61 microscope (Olympus Corporation) and analyzed by CytoVision software (Leica Biosystems). An average banding resolution of 300 bands was achieved. Aberrations were described according to the International System for Human Cytogenetic Nomenclature, 2016 (ref. ⁵⁸ ).