2.4.1. Single Cell Gel Electrophoresis Assay (Comet Assay)

Comet assay was conducted as described previously [43] with few modifications. Specifically, 3 × 105 HaCat cells were seeded and cultured on 60 mm plates 24 h prior to the experiment. Cells were then washed with Phosphated buffer saline (PBS) (w/o Ca²⁺/Mg²⁺) (Biosera) and irradiated with 55 mJ/cm² UVB (12 s) with a Bio-Link BLX254 Crosslinker (in PBS) (Vilber Lourmat, Marne-la-Vallée, France). Cells were allowed to recover for 16 h either with culture medium alone or with the culture medium supplemented with 0.1% v/v CRPP. As a control, a group of non-irradiated cells was also included in the experiment.

Subsequently cells were collected through trypsinization, washed and diluted in ice-cold PBS (2 × 104 cells/mL of PBS w/o Ca²⁺/Mg²⁺) and 0.4 mL of the diluted cells were then suspended in 1.2 mL low-gelling-temperature agarose (1% (w/v) in ddH₂O) and coated to super frosted glass microscope slides precoated with a layer of 1% low-melting point agarose (warmed to 37 °C prior to use) and allowed to set for at least 2 min at RT. Cells were then lysed in lysis solution (1.2 M NaCl, 100 mM Na₂EDTA, 0.1% sodium lauryl sarcosinate and 0.26 M NaOH, pH = 13) for 1 h at 4 °C in the dark to achieve the denaturation of the two DNA strands (alkaline unwinding). Following lysis, slides were washed twice with rinse solution (0.03 M NaOH and 2 mM Na₂EDTA, pH~12.3) for 20 min at RT, subjected to electrophoresis for 25 min (in the presence of rinse solution) at 0.6 V per cm between the positive and negative electrodes of the electrophoresis apparatus. Slides, were subsequently neutralized in ddH₂O, stained with propidium iodide (10 μg/mL in ddH₂O) for 20 min and processed for observation on a Nikon ECLIPSE E200 fluorescence microscope.

For the estimation of DNA damage 100 comets of each slide were scored as class 0 to 4 in proportion to the observed tail due to DNA damage, observed “tail” as described previously (Collins A. et al. 1997). Each comet was assigned a value on the basis of its class and the overall score of 100 comets ranged from 400 (100% comets of class 4) to 0 (100% comets being in class 0). In this way, the overall DNA damage of the sample was expressed in arbitrary units.