CometChip assay

DNA strand breaks were assessed using the CometChip Platform (Trevigen).³⁹ MEF cells were seeded in 96-well plate at a density of 10⁴ cells per well. The next day, cells were untreated, treated with 150 μM BPA, 80 nM CPT, or co-exposed by treating with 150 μM BPA for 1 h followed by 24 h and 48 h exposure with 80 nM CPT and 150 μM BPA. After the designated exposure periods, cells were trypsinized and moved into the chilled 30-micron sized CometChip® apparatus. Cells were gravity loaded into the microwells for 30 min with the CometChip® placed in the cold room (4°C). After loading in the CometChip® apparatus, the chip was washed multiple times with PBS and sealed with low melting point agarose (LMPA, ThermoFisher) (7 ml; 0.8% LMPA/ PBS). The CometChip® was then submerged in lysis solution with detergent for 40 min at 4°C. The CometChip® was run under alkaline (pH>13) conditions (200 mM NaOH, 1 mM EDTA, 0.1% Triton X-100). Electrophoresis was conducted 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). Subsequently, the DNA was stained with 1x SYBR® Gold diluted in Tris buffer (20 mM Tris·Cl, pH 7.4) for 30 min and de-stained for 1 h in Tris buffer (20 mM Tris·Cl, pH 7.4).

Image acquisition was conducted on the Celigo S imaging cytometer at a resolution of 1 micron/pixel with whole plate imaging to avoid imaging variability. Image analysis was conducted using the dedicated Trevigen Comet Analysis Software (CAS), with the box size set to 220 × 180 pixels which represented a box size that would capture comets from heavily damaged cells without box overlap. For all data, % tail DNA was used to measure the amount of DNA damage, which was calculated as the amount of tail DNA to the total amount of DNA multiply by 100. Data of at least four technical replicates, each with 1500 ± 300 comets were acquired and exported to Excel and subsequently to Graphpad for statistical analysis. Detailed analysis of the CometChip Platform has been described³⁹.