Mitotic Chromosome Preparation from Blood

The mitotic chromosomal suspensions were prepared by leucocyte cultivation from fresh peripheral blood. Our protocol was previously described in Mazzoleni et al. [32 (link)]. Briefly, the cultivation medium (100 mL) was prepared by using 90 mL of commercially prepared D-MEM medium (Sigma-Aldrich, St. Louis, MO, USA), enriched with 10 mL of fetal bovine serum (Gibco, Carlsbad, CA, USA), 3 mL of phytohemagglutinin M (Gibco, Carlsbad, CA, USA), 1 mL of penicillin/streptomycin solution (10000 units/mL; Gibco, Carlsbad, CA, USA), 1 mL L-glutamine solution (200 mM; Sigma-Aldrich, St. Louis, MO, USA), and 1 mL lipopolysaccharide solution (10 mg/mL; Sigma-Aldrich, St. Louis, MO, USA). Then, 100–200 μL of blood were cultivated in 5 mL of the medium for one week at 30 °C. Afterwards, 35 μL colcemid (Roche, Basel, Switzerland) was added 3.5 h before harvesting. The cells were hypotonized in pre-warmed 0.075 M KCl solution for 30 min at 30 °C. Subsequently, the cells were washed four times and stored in Carnoy’s fixative solution (methanol: acetic acid, 3:1).
Chromosomal preparations were dropped to slides and stained by Giemsa for karyotype reconstruction and C-banding for visualization of constitutive heterochromatin, following the protocol of Sumner [33 (link)] modified according to Mazzoleni et al. [32 (link)].