Chromosome Preparation in Madagascar Leaf-tail Geckos

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.