3.5. Comet Assay for Genotoxicity Testing

Human fibroblasts were used for this study. Cells were cultured on 6-well plates at 37 °C, 5% CO₂ in DMEM (Gibco) supplemented with 10% FBS (Gibco) and penicillin/streptomycin (Gibco), for 72 h. Subsequently, the confluent cells were incubated for 24 h with test concentrations (v/v) of the oil (50, 100, 150 and 200 mg/g). As a positive control, cells incubated with 100 μM of H₂O₂ were used. Afterward, the medium was removed, and the cells were trypsinized, suspended in fresh medium and centrifuged. Next, 10 μL of the cell pellet was resuspended in 75 μL of a 0.5% low melting agarose solution (LMPA). Thereafter, the cell suspension was applied to the cooled primary slides coated with 1% regular agarose solution (NMA) and covered with a coverslip for approximately 5 min. After that step, the coverslip was detached, and the third LMPA agarose layer was applied. Afterwards, the cells were lysed for 60 min in lysis buffer at 4 °C (freshly made) (2.5 mol/L NaCl, 10 mmol/L Tris, pH 10; 1% sodium sarcosinate, 10% dimethylsulfoxide, 100 mmol/L EDTA, and 1% Triton X-100 (Sigma Chemical Laboratories)) without access to light and incubated for 20 min in an electrophoresis buffer (300 mmol/L NaOH, 1 mmol/L EDTA; pH 13). Electrophoresis was conducted at 300 mA and25 V. Afterwards, the slides were rinsed three times in the neutralizing buffer, stained with ethidium bromide (Sigma-Aldrich), observed with a fluorescence microscope. The obtained photos were analyzed by applying the CometAssay Lite IV software (π Perceptive Instruments, UK). Next, 50 cells were analyzed per sample point. Comet tail moment was expressed as a percent of the control value.