Phytohemagglutinin m

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.

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)].

Chromosome suspensions were prepared from whole blood cell cultures. Briefly, the cultivation medium consisted of 90 mL of 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 (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). Then, 100–300 μL of blood samples were added to the 5 mL of cultivation medium and incubated for one week at 30 °C, without CO₂ supplementation. After the incubation period, the cultures were first treated with 35 μL of colcemid (10 μg/mL; Roche, Basel, Switzerland) and incubated for 3 h and 30 min at 30 °C. The cells were treated with a pre-warmed 0.075 M KCl hypotonic solution for 30 min at 37 °C, washed and fixed four times with cold 3:1 methanol/acetic acid solution. The chromosome suspensions were later stored at −20 °C for further use.

RSV, 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA), tert-butylhydroperoxide (t-BHP), methanol, ethanol, dimethyl sulfoxide, Triton X-100, sodium hydroxide, sodium chloride, ethylenediamine tetraacetic acid (EDTA) agarose for routine, agarose low electroendoosmosis (EEO), hydrogen peroxide (H₂O₂), hypoxanthine, ethidium bromide, methylene blue (MET), xanthine oxidase and cytochrome C were purchased from Sigma Chemical Com. (St. Lois, MO, USA). DDS hydroxylamine was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). phytohemagglutinin M. was purchased from Life Technologies (Carlsbad, CA).

Racemic Lipoic Acid (ALA), R-Lipoic Acid (R-ALA) and S-Lipoic Acid (S-ALA),2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), tert-butyl hydroperoxide (t-BHP), methanol, ethanol, dimethyl sulfoxide, Triton X-100, sodium hydroxide, sodium chloride, ethylenediamine tetraacetic acid (EDTA) agarose for routine, agarose low electroendosmosis (EEO), hydrogen peroxide (H₂O₂), hypoxanthine, ethidium bromide, methylene blue (MET), xanthine oxidase and cytochrome C were purchased from Sigma Chemical Com. (St. Louis, MO, USA). Dapsone hydroxylamine (DDS-NOH) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phytohemagglutinin M. was purchased from Life Technologies (Carlsbad, CA, USA).