2.4.7. Fast Halo Assay

The fast halo assay (FHA) was conducted to evaluate the effect of the tested oils on double-strand breaks (DSBs). Cells were seeded on 24-well plates at a density 2.5 × 10⁴ cells/ml per well. After incubating with the tested oils, the supernatant was transferred to centrifuge tubes, which were previously properly signed and prepared, and the culture was washed with PBS, which was also collected into tubes. The cells were separated from the surface of the plate with TrypLE solution up to 3 min at 37°C in a CO₂ incubator, and the cell suspensions were transferred into tubes, which were then centrifuged at 1000 × g for 5 min. After removal of the supernatant, the cell pellet was washed in PBS and again centrifuged at 1000 × g for 5 min. The cells were resuspended in PBS with Ca²⁺ and Mg²⁺ at a density of 1000 cells/μl; the cells in tubes were placed in a water bath. Then, 120 μl of 1.25% low melting agarose in PBS was added to the cells, and the mixture was immediately squeezed between a slide coated with agarose (high melting point) and a coverslip. The slides were placed on a cooling block for 10 min for gel formation. The coverslips were then removed, and the slides were placed in the lysis buffer overnight. The slides were then transferred into alkaline solution (pH = 13.0) for 30 min and then washed twice for 5 min in neutralizing buffer. The preparations were stained using 5 μM DAPI for 20 min and examined immediately under a fluorescence microscope. Photographs were taken, and then, the ratio of cell nucleus diameter to halo diameter (which is a measure of DNA damage) was analyzed [29].