Vista green dna dye

Comet slides were pre-coated with 1% normal-melting-point agarose and dried at 60 °C. After their exposure to 1,2-DCP, the cells were washed in cold PBS, centrifuged, and then suspended in 0.75% low-melting-point agarose at a ratio of 1:10 (v/v) in a single cell suspension. Next, 100 µL of the single cell suspension was pipetted onto the comet slides and kept at 4 °C in the dark for 20 min. The cells were then treated with pre-chilled lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, pH > 10, 1% N-lauroylsarcosine, 1% Triton X-100, 10% DMSO) at 4 °C for 1 h and then in pre-chilled alkaline solution (300 mM NaOH, pH > 13, 1 mM EDTA) for 30 min at 4 °C in the dark. Alkaline electrophoresis was performed at 17V with current set at 200 mA for 15 min. The slides were then washed thrice in pre-chilled distilled water, fixed in 70% ethanol, dried at 37 °C in the dark, and then stained with vista green DNA dye (Cell Biolabs, Inc., San Diego, CA, USA) for 15 min at room temperature. The slides were rinsed briefly in distilled water and dried at 37 °C for an hour as described previously [46 (link)]. They were examined under a fluorescent microscope (Leica DMI6000B-AFC, Wetzlar) and images were taken. At least 100 cells per slide were analyzed using Comet Score V2.0 (TriTek Corp., Sumerduck, VA, USA). The positive control cells were exposed to 0.2 mM of H₂O₂ for 2 h.

The comet assay was performed under alkaline conditions using the OxiSelect^TM Comet assay kit (Cell Biolabs Inc. San Diego, CA, USA) following the manufacturer’s instructions. Briefly, electrophoresis was carried out on 1 × 10⁵ cells layered on comet slides for 30 min at 25 V and 300 mA. Then the slides were stained with 100 μL/well of diluted Vista Green DNA Dye (Cell Biolabs Inc.). The comet images were captured with an Olympus IX71 fluorescence microscope (200X magnification).

Comet assays were performed using the OxiSelect Comet Assay Slides according to the manufacturer’s instructions (Cell Biolabs Inc., San Diego, CA). A cellular suspension in PBS (1 × 10⁵ cells per mL) was mixed 1:10 with molten low-melting-point agarose at 37 °C. Seventy-five microliters of this suspension were then placed within each well on the specially prepared microscope slide provided with the kit. Slides were incubated at 4 °C for 15 min to allow the agarose to solidify and then were placed in lysis buffer and incubated for 30 min at 4 °C in the dark. Denaturation was achieved by transferring the slides to a pre-chilled alkaline electrophoresis buffer (300 mM sodium hydroxide, 1.0 mM EDTA) and incubating for 30 min at 4 °C in the dark. The slides were then transferred into a horizontal electrophoresis chamber (BioRad Inc., Hercules, CA) and subjected to electrophoresis at 1 V/cm (30 V, 300 mA) for 15 min. Following electrophoresis, slides were washed 3 times with water, once with 70% ethanol, and air dried. The dried slides were stained with 100 μL of diluted Vista Green DNA dye (Cell Biolabs Inc., San Diego, CA), and cellular DNA was visualized using a FITC filter-fitted microscope. Comet images were scored visually on a scale of 0 to 4 as described by Collins [23 (link)], with 0 representing no damage and 4 representing severe damage.

Transfected cells and their controls subjected to radiation, or not, were placed on ice immediately after x-ray exposure. Cells were collected, counted, and 2×10⁴ cells were mixed with 1% solution of molten low melting agarose at 1:10 ratio. Cell-agarose complexes were transferred onto agarose-coated glass slides, allowed to solidify, kept at 4 °C for 15 min, and submerged into pre-chilled neutral lysis solution for DSB detection containing 2% sarkosyl, 0.5 M disodium ethylenediaminetetraacetate dihydrate (Na₂EDTA-2H₂O), 0.5 mg/ml proteinase K (pH 8.0) for 18 h at 37 °C. Agarose slides were submerged into running buffer containing 90 mM Tris, pH 8.5, 90 mM boric acid, 2 mM Na₂EDTA-2H₂O for 30 min with two additional washes followed by DNA electrophoresis at 1 V per cm, wash in deionized H₂O and ice-cold 70% ethanol, air dried and stained with 1:10,000 dilution of Vista Green DNA Dye (Cell Biolabs, San Diego, CA) for 15 min. Slides were imaged using Zeiss Axiovert fluorescence microscope. At least ten random fields were imaged per each slide. Fifty comets per slide were analyzed using CaspLab.com software.

Dulbecco’s modified Eagle’s medium (DMEM), Fetal bovine serum (FBS), MTT reagent, Dead green viability stain, γH2AX primary antibody, Alexa Fluor-555 conjugated secondary antibody, Lipofectamine® 3000 Reagent were procured from Invitrogen, USA. Vista green DNA dye and MMP-2 primary antibody was procured from Cell Biolabs, USA and Santa Cruz Biotechnology respectively.

DNA damage in single cells was determined with a gel electrophoresis assay (the Comet assay). The alkaline Comet assay detecting DNA with both single-strand breaks (SSBs) and double-strand breaks (DSBs) [34 (link), 35 (link)] was performed using the OxiSelect^™ Comet assay kits following the manufacture’s instruction. After the staining with Vista Green DNA Dye (Cell Biolabs), the slides were examined under an Olympus fluorescent microscopy and images were captured. Images of the Comet assay from two experiments with 60–100 cells each treatment group were analyzed using OpenComet (v1.3.1), a plugin [36 (link)] for the imaging processing software Image J, to automatically calculate Tail DNA% (= 100 x Tail DNA Intensity/DNA Intensity) and Extent Tail Moment (= Length of Tail x Tail DNA%).