Evaluating γH2AX as a DNA Damage Marker

γH2AX was evaluated as a marker for DNA double-strand breaks (DDS). The phosphorylation of the histone H2AX is in fact related to the formation of DDS in response to several toxicant, oxidative stress and after cell cycle arrest [27 (link),28 (link)] and γH2AX has been proposed as the most informative marker of double-strand breaks [29 ]. A549 cells were seeded (2.5 × 10⁵ cells/well) in a 6-well cell culture plate and incubated overnight. Cells were treated with 20 or 50 µg/mL of silver NPs for 24 h or with etoposide (1.65 µM) as a positive control. At the end of the treatment, cells were washed with PBS, collected by centrifugation, fixed using 4% PFA for 15 min and permeabilized with ice-cold 90/10% methanol/PBS for 10 min. The samples were stained using Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (Alexa Fluor^® 488 Conjugate) (Cell Signaling Technology, Danvers, MA, USA) following the manufacturer instructions. γH2AX fluorescence intensity was measured using flow cytometry (CytoFlex, Beckman Coulter, Cassina de Pecchi, Italy). Fluorescence was measured immediately using an excitation wavelength of 488 nm and an emission wavelength of 525 nm and measuring 10,000 events for each sample.