Chromosome Suspension Preparation from Blood

Chromosome suspensions were obtained from whole-blood cell cultures [71 (link)]. The medium used for the cultures consisted of 90 mL D-MEM cell culture medium (without glucose, L-glutamine and sodium pyruvate; GIBCO, Carlsbad, CA, USA), 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 (10,000 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). Approximately 100–200 μL of blood was added to 5 mL of fresh medium and incubated for one week at 30 °C. After the incubation period, the mitotic cycle was arrested in metaphase by adding 35 μL of colcemid solution (10 μg/mL; Roche, Basel, Switzerland) to each sample. Following an incubation period of 3.5 h at 30 °C, the samples were centrifuged at 1200 rpm for 10 min at room temperature and incubated with 0.075 M of prewarmed KCl for 10 min at 37 °C. The blood cells were centrifuged again at 1200 rpm for 10 min at 4 °C, resuspended with 5 mL of fixative (3:1 methanol:acetic acid) and incubated for 20 min at 4 °C. The last step was repeated two additional times for better fixation, and the chromosome suspensions were stored at −20 °C until further use.