*4.7. DNA Double-Strand Breaks Assessment*

A fast halo assay (FHA) [62] was performed to assess DSBs in the DNA of differentiated PC12 cells treated with glucose or insulin. This test enables the rapid assessment of the extent of DNA breakage caused by different types of DNA lesions. After 1 h and 24 h incubation with glucose or insulin, the supernatants were collected into tubes. The trypsinization process was performed with a TrypLE solution (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for 3–5 min in a CO2-incubator (37 ◦C, 5% CO2, 95% humidity). After detaching from surfaces, suspended cells were collected into tubes and centrifuged

for 5 min at 1000× *g* (Eppendorf, Hamburg, Germany) to get rid of cellular debris. Next, the supernatant was removed, and the cell pellet was washed with PBS and centrifuged again under the same conditions. The cells were then re-suspended at the density of 1000 cells/μL in sterile PBS and put in bathwater (37 ◦C). Then, 120 μl of 1.25% agarose (low melting point) in sterile PBS was added to cells and immediately sandwiched between an agarose-coated (high melting point) slide and a coverslip. After complete gelling (cooling block for 10 min), the coverslips were removed, and the slides were placed into the lysis buffer overnight at 4 ◦C in the dark. The next day, the slides were transferred into a Tris-HCl buffer (pH = 13) for 30 min in the dark and then twice into a neutralization buffer for 5 min. Finally, the slides were stained using 5 μM of 4 ,6-diamidino-2-phenylindole (DAPI) for 20 min and analyzed under a fluorescence microscope. The DAPI-labelled DNA was visualized using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany), and the subsequent images were digitally recorded on a PC and analyzed with imageanalysis software developed by one of co-authors. The slides were numerically coded before reading to reduce operator bias. The extent of strand scission was quantified by calculating the nuclear diffusion factor (NDF), which represents the ratio between the total area of the halo plus nucleus and that of the nucleus. Data are expressed as relative NDF, calculated by subtracting the NDF of control cells from that of treated cells. All experiments were performed at least 3 times.
