Comet Assay for Asbestos-Induced DNA Damage

Exponentially growing human pleural mesothelial cells were cultured in 6-well plates and exposed to 50 µM LGM2605 4 h prior to exposure to sterile crocidolite asbestos fibers (20 µg/cm²). As previously described [20 (link)], cells were processed for Comet assay as per manufacturer’s instructions (Trevigen, Gaithersburg, MD, USA). Briefly, harvested mesothelial cells (1 × 10⁵ cells/mL in 1× PBS) were mixed with LMAgarose^® (1:10, v/v) and immediately pipetted onto CometSlides™. Cells were then lysed (4 °C for 30 min) and kept in the dark for unwinding (RT). Electrophoresis was done in a horizontal electrophoresis unit at 18 volts (200 Amp) for 25 min. Slides were washed twice with distilled water, fixed in 70% ethanol and dried at 45 °C. DNA was stained by SYBR green (Trevigen, Gaithersburg, MD, USA). At least 100 cells were scored per group. Visual analysis of cells and comet tail length was measured using Comet Image Analysis software (Comet assay IV, Perceptive Instruments Ltd., Haverhill, MA, USA). Images were captured on an Olympus IX51 fluorescence microscope using a monochrome CCD FireWire camera with a 40× objective lens.