Gurr buffer

U2OS were incubated with 240 μM BrdU for two cell cycles and treated with BPDE, 4-NQO, and siRNA as indicated. 4 h before trypsinization, 0.02 μg/mL colcemid (Sigma-Aldrich) was added. In total 0.075 M KCl hypotonic buffer was added to pelleted cells for 15 min and cells were then fixed in 3:1 methanol:acetic acid. Spreads were prepared by dropwise addition to a glass slide and left to dry in the dark overnight. Slides with metaphases were stained in 10 μg/mL Hoechst 33258 in water for 20 min, exposed to long-wave (365 nm) UV light in a glass dish of 2x SSC buffer (30 mM sodium citrate, 300 mM NaCl in water) for 8–10 min, and stained in 20% Leishman’s stain (VWR) diluted in Gurr buffer (Gibco) for 6 min at room temperature. Slides were analyzed using a Zeiss Axio Observer microscope. SCEs were defined by an exchange of dark-stained (Hoechst-bound TT-rich) segments with light, bleached (BrdU incorporated) segments. Staining variation where chromatids clearly twisted around each other was excluded from the count. For each condition, at least 90 metaphases from at least 3 independent biological repeats were analyzed. SCE rates were calculated per diploid set of chromosomes to compensate although U2OS present with chromosome counts in the hypertriploid range. SCEs were quantified directly on the microscope, and representative images were taken for illustration.

Bleomycin- or MTX-treated cells were exposed to colchicine (Sigma-Aldrich) at a final concentration of 10 μg/mL for 2 h, trypsinized, washed with phosphate-buffered saline (PBS; Life Technologies), and treated with 0.56% KCl at room temperature for 10 min. The cells were then fixed with a 3:1 (v/v) mixture of methanol: acetic acid at −20 °C for 5 min and spread onto a glass slide. The cell spreads were baked at 65 °C for 16 h, treated with 0.025% trypsin in PBS for 80–100 s, washed with 0.9% NaCl in H₂O, stained with 4% Giemsa solution in Gurr buffer (Gibco) for 10 min, and imaged using a 100× oil immersion objective on an EVOS^® XL microscope (Life Technologies). The karyotypes were compared to the standard karyotype of CHO-DUK cells [18 (link)] to identify chromosomal rearrangements.

SKY was performed using SKY Paint kit-Human (Applied Spectral Imaging) as per manufacturer’s protocol. The samples were then analyzed using the D300 bio imaging system (Applied Spectral Imaging). For chromosome spreading, cultured cells were treated with Colchicin (10 μg/ml for 6 h). Cells were then collected, and the suspended cells were treated with hypotonic solution (KCl) and fixed with Methanol and Acetic Acid (3:1 ratio). Chromosome spreading and Giemsa staining (4% in Gurr buffer for 2 h) (Gibco) were performed by conventional methods.

Briefly, blood from a female animal (NN03) was collected in a sodium-heparin vacutainer (Becton–Dickinson) and used for short-term (72-h) lymphocyte cultures as previously described^{89 (link)}. We used phytohemagglutinin (PHA from Phaseolus vulgaris, 20 μg ml^–1, Sigma Aldrich) as the mitogen. Additionally, we established primary fibroblast cultures under sterile conditions using small pieces (0.5 mm²) of ovarian tissue from NN03. The fibroblasts were incubated in MEM alpha containing nucleosides and GlutaMax (Thermo Fisher), supplemented with 20% fetal bovine serum (Atlanta Biologicals) and antibiotic-antimycotic (Thermo Fisher) at 30 °C with 5% CO₂. Metaphase chromosomes were obtained from both lymphocyte and fibroblast cultures by arresting cells with demecolcine (KaryoMax, Thermo Fisher; final concentration, 0.1 μg ml^–1), followed by hypotonic treatment with Optimal Hypotonic Solution (Rainbow Scientific) and fixation in methanol/acetic acid (3/1). Metaphase spreads were prepared on precleaned wet glass slides at room temperature. Chromosomes were stained with 5% Giemsa (KaryoMax, Thermo Fisher) in GURR buffer (Gibco). At least 30 metaphase spreads were captured and analyzed for karyotyping using an Axioplan2 microscope (Zeiss) and Ikaros (MetaSystems) software.

The cells were seeded in culture flasks to stimulate proliferation. After treatment with colcemid (4-6 h at 0.15 μg/mL) the cells were detached using trypsin and concentrated by centrifugation. The cells were treated with hypotonic solution (75 mM KCl, 3 min), concentrated again and fixed with 10 volumes fixative 1 [methanol-acetic acid 9:1 (v/v)]. The cells were concentrated and subsequently fixed with 10 volumes fixative 2 [methanol-acetic acid 3:1 (v/v)]. The fixed cell suspension was concentrated once more after which the cells were dropped onto microscope slides. The slides were allowed to dry and aged overnight at 60 °C. The slides were re-hydrated in 2× SSC and subsequently in PBS, treated with trypsin (0.07%, 3 min) and finally stained at room temperature for 3–4 min according to Giemsa-Leishman (G-banding) using 1 ml Giemsa (Merck) and 3 mL Leishman stain (Merck) in 60 ml Gurr buffer (Gibco). The slides were dried and mounted in Eukitt^®.