Fig 1: XPTx-0091 increases MMS-induced DNA damage. (A) The alkaline CometChip assay was used to quantify the amount of DNA damage in XPTx-0091-treated LN428/MPG cells in the presence or absence of MMS. (B) Low-level MPG expressing LN428 cells were also tested using the CometChip assay. In brief, cells were gravity-loaded into 30 µM microwells of the CometChip apparatus for 30 min at 4 °C. After loading, the wells were washed multiple times with PBS and sealed with 0.8% low-melting-point agarose (Topvision, Thermo Fisher Scientific, Waltham, MA, USA, Cat# R0801) in PBS. The CometChip was then submerged in lysis solution with Triton X-100 detergent (BioTechne, Minneapolis, MN, USA, Cat# 4250-050-01) for 2 h at 4 °C and subsequently electrophoresed under alkaline (pH > 13) conditions (200 mM NaOH, 1 mM EDTA, 0.1% Triton X-100) at 22 V for 50 min at 4 °C. After electrophoresis, the CometChip was re-equilibrated to neutral pH using Tris buffer (0.4 M Tris·Cl, pH 7.4), and the DNA was then stained with 1x SYBR Gold (Thermo Fisher Scientific, Cat# S11494), diluted in Tris buffer* (20 mM Tris·Cl, pH 7.4) for 30 min, and de-stained for 1 h in Tris buffer*. Image acquisition was conducted on a Celigo S imaging cytometer (Nexcelom Bioscience, Lawrence, MA, USA) at a resolution of 1 micron/pixel with whole plate imaging to avoid imaging variability. Image analysis was conducted using the dedicated Comet Analysis Software (CAS) v 1.2 (https://casplab-comet-assay-software-project.soft112.com (accessed on 20 October 2023)) with the box size set to 220 × 180 pixels, representing a box size that would capture comets from heavily damaged cells without box overlap. The data acquired were exported to Excel (Microsoft, Redmond, WA, USA) and subsequently to GraphPad (Prism, V9) for statistical analysis. Data are shown as mean +/− SEM; *** p < 0.001.
Supplier Page from R&D Systems, a Bio-Techne Brand for CometAssay Lysis Solution (2 x 500 mL)