4.4. Fpg-Modified Alkaline Comet Assay

The Fpg-modified alkaline comet assay was performed as described by Xiao et al. [42] with slight modification. Cells with and without treatment were collected, washed with PBS, and suspended in 0.6% low melting point agarose (LMPA) at an appropriate concentration and placed onto slides. Slides were incubated in lysis buffer (100 mM Ethylenediaminetetraacetic acid (EDTA), 2.5 M NaCl, 10 mM, Tris-HCl, 1% Triton-X; pH 10) overnight at 4 °C and equilibrated in enzyme reaction buffer (40 mM HEPES, 0.1 M KCl, 0.6 mM EDTA, 0.2 mg/mL Bovine serum albumin (BSA); pH 8). Then, samples were either treated with 8 U/mL of Fpg (M0240S, NEB, Ipswich, MA, USA) in NEB buffer1 or in buffer1 without Fpg for 30 min at 37 °C. After separating DNA strands in alkaline buffer (0.3 M NaOH, 1 mM EDTA) for 20 min at 30 V and 300 mA, we placed slides in neutralization buffer (0.4 M Tris-HCl; pH 7.5) for 20 min to allow for DNA recondensation. Samples were dried overnight and stained with propidium iodide at 2 µg/mL. Slides were imaged using a Nikon Eclipse at 20X objective, and tail moment was calculated using OpenComet plugin for ImageJ. More than 50 cells were analyzed for each sample and the average tail moment with 95% CI was calculated.