Colcemid

Contact inhibited G0/G1 phase AG1522 cells were pretreated with SAHA for 24 h before γ-ray irradiation. Immediately after irradiation, cells were trypsinized and replated. The first post irradiation metaphase cells were harvested with 0.1 μg/mL Colcemid (Invitrogen Life Technologies) treatment and prepared as a standard method [57 (link)]. Samples were treated in 75 mM KCl solution for 20 min at 37 °C and fixed in 3:1 (methanol: acetic acid) Carnoy fixation solution three times. Metaphase spreads were stained with 5% Giemsa solution, and the chromosome aberrations (dicentric chromosomes and ring chromosomes) were analyzed. A minimum of 100 metaphase cells was analyzed for each data point for at least two independent experiments.

For metaphase spread preparation, cells were harvested 20 hours after irradiation. Colcemid (Invitrogen) was added at a final concentration of 0.1 µg/mL 2 hours prior to harvesting. Cells were trypsinised (0.05% trypsin, Hyclone) for 1–2 min at room temperature, the trypsin was inactivated with foetal bovine serum (Hyclone), and a cell suspension was prepared. Hypotonic treatment was performed with 0.075 M KCI at 37°C for 10 min. Thereafter, the cells were fixed with methanol-acetic acid fixative following a standard protocol, modified as described previously18 (link). The slides were stained with Giemsa stain. They were coded prior to analysis, and chromosomal aberrations were scored in a blinded manner. At least 200 euploid metaphases were analysed per sample for the presence of chromosome-type and chromatid-type aberrations. A two-sided exact Fisher's test was used to determine the statistical significance of the differences (p<0.05).

The cells were incubated in ESCs medium with 0.25 μg/ml colcemid (Invitrogen, Thermo Fisher Scientific) for 2-3 h and harvested with 0.05% Trypsin-EDTA (Invitrogen, Thermo Fisher Scientific). After incubation in hypotonic solution containing 0.4% sodium citrate and 0.4% potassium chloride (1:1, v/v) at 37°C for 5 min, the cells were fixed with a methanol/acetic acid mixture (3:1, v/v). The fixed cells were mounted on glass slides and stained with Giemsa at 37°C for 10-15 min after drying. At least 20 metaphase chromosome karyoschisis were examined for each cell line.

Cells were treated with 0.5 μg/ml of Colcemid (Invitrogen) for 4 h before harvest. Chromosomes were fixed with 3% formamide and hybridized with a telomere PNA-FISH 5′-Cy3-OO-(CCCTAA)₃–3′ probe (PNAgene) as described in Wu et al. (10 (link)). DNA was counterstained with DAPI. Digital images were captured using NIS-Elements BR (Nikon) with a Nikon Eclipse 80i microscope utilizing an Andor CCD camera. The relative telomere signals were analyzed with ImageJ software (downloaded from Fiji). For CO-FISH, cells were incubated with 10 μM BrdU for 12 h, treated with 0.5 μg/ml of Colcemid for 4 h and harvested. Formalin-fixed metaphase spreads were stained with 0.5 μg/ml of Hoechst 33258 (Sigma) in 2 × SSC for 15 min at room temperature before being exposed to UV light equivalent to 5.4 × 10³ J/m². After digestion with 200 U of Exonuclease III (Promega), the samples were denatured at 85°C for 3 min and incubated sequentially with 5′-Cy3-OO-(CCCTAA)₃–3′ and 5′-FAM-OO-(TTAGGG)₃–3′ probes as described above. Images were captured as described above.

CHO cells were synchronized into the G1 phase using a mitotic shake-off procedure [28 (link),29 (link)]. Synchronized mitotic cells were subcultured in 6 well plates and incubated for two hours at 37 °C. Cells were then treated with various concentrations of testing chemicals and incubated with 10 µM of BrdU (Sigma, St Louis, MO, USA) for two cell cycles. Then 0.2 µg/mL of colcemid (Gibco, Invitrogen, Grand Island, NY, USA) was added to cells and allowed to incubate for an additional 6 h. Cells were harvested during metaphase, trypsinized, and then suspended in 2 mL of 75 mM KCl solution warmed to 37 °C and placed in a 37 °C water bath for 20 min. A fixative solution of 3:1 methanol to acetic acid was added to the samples according to the standard protocol [30 (link)]. Fixed cells were dropped onto slides and allowed to dry at room temperature. Differential staining of metaphase chromosomes was completed using the fluorescence plus Giemsa technique with Hoechst 33258 dye [31 (link)]. Differentially stained metaphase chromosome images were scored under a Zeiss Axioskop microscope equipped with a SPOT CCD camera RT 2.3.1 (Diagnostic Instrument, Inc., Sterling Heights, MI, USA) and SPOT basic software. A minimum of 50 metaphase cells were scored for each treatment concentration. Data presented are the mean of SCE frequency per chromosome.