Colcemid solution

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.

Bone marrow from the tibias of each bird was flushed out using a syringe needle with D-MEM medium (Sigma Aldrich) and cultivated in 5 ml of D-MEM medium (Sigma Aldrich) with 75 µl of colcemid solution (Roche) for 40 min at 37°C. After that, the cells were hypotonized in pre-warmed 0.075 M KCl solution for 25 min at 37°C. Finally, cells were washed four times with fixative solution (methanol:acetic acid, 3:1) and then stored at −20°C prior to use.
Chromosomal spreading was done using the air-drying technique followed by conventional Giemsa staining (5% Giemsa in 0.07 M phosphate buffer, pH 7.4). The C-banding method was applied for visualization of constitutive heterochromatin according to Sumner (1972) (link). More specifically slides with chromosomal spreads were aged at 60°C for 1 h then successively soaked in 0.2 N HCl for 20 min at room temperature then in 5% Ba(OH)₂ solution for 4–5 min at 45°C and subsequently in 2× SSC for 1 h at 60°C, with intermediate washes in distilled water. Finally, metaphases were mounted with 4′,6-diamidino-2-phenylindole (DAPI) in mounting medium Vectashield (Vector laboratories).

Chromosome suspensions were prepared from whole-blood cell cultures in three species of the Madagascar leaf-tail geckos (U. alluaudi, U. henkeli, U. sikorae), following the protocol described in [45 (link)]. Briefly, the culture medium consisted of 90 mL of DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) enriched with 10 mL of fetal bovine serum (GIBCO), 3 mL of phytohemagglutinin M (GIBCO), 1 mL of penicillin/streptomycin solution (10,000 units/mL; GIBCO, Waltham, MA, USA), 1 mL of L-glutamine solution (200 mM; Sigma-Aldrich) and 1 mL of lipopolysaccharide solution (10 mg/mL; Sigma-Aldrich). Subsequently, 100–300 μL of blood was added to 5 mL of cultivation medium and incubated at 30 °C for one week. After the incubation period, we used 35 μL of colcemid solution (10 μg/mL; Roche, Basel, Switzerland) to block cell division and then incubated the cultures for 3 h and 30 min at 30 °C. Subsequently, the cells were treated with a pre-warmed hypotonic solution (0.075 M KCl) for 30 min at 37 °C, washed by centrifugation at 800–1200 rpm for 10 min, and fixed four times with cold 3:1 methanol/acetic acid solution for 20 min each, with intermediate centrifugation at 1200 rpm for 10 min. Chromosome suspensions were spread onto slides and incubated at 60 °C for 1 h, prior to all cytogenetic experiments. The remaining chromosome suspensions were stored at −20 °C.