Fluorescein isothiocyanate antiphosphorylated ser139 h2ax clone 2f3

PBMCs were stained with Pacific Blue anti-human CD3, APC anti-human CD4, PE anti-human CD8a and APC/Cy7 anti-human CD14 (Bio Legend, San Diego, California, USA). The stained cells were then fixed and permeabilised using the Nuclear Factor Fixation and Permeabilization Buffer Set (BioLegend, San Diego, California, USA) as per the manufacturer's instructions. Intracellular staining was then performed using fluorescein isothiocyanate anti-phosphorylated (ser139) H2AX (clone 2F3; BioLegend). For the cell-cycle experiments, the PBMCs were additionally treated with 20 μg/mL RNase A (Sigma-Aldrich, St. Louis, Missouri, USA), and then stained with 40 μg/mL propidium iodide (BioLegend) as per manufacturer's instructions for 1 hour prior to analysis. Flow cytometry analysis was then performed using a MoFlow Astrios Flow Cytometer and Summit software V.6.2.3 (Beckman Coulter, Miami, Florida, USA). Phospho-H2AX levels are reported as median fluorescence intensity (MFI) values.