Alexa fluor 488 conjugated goat anti mouse f ab 2 fragments

To assess γH2AX foci formation by immunofluorescence microscopy, cells were cytocentrifuged, air-dried and fixed in 4% formaldehyde, followed by autofluorescence quenching in 50 mM NH₄Cl, permeabilization with 0.2% Triton-X and incubation for 1 hr at room temperature with a mouse-anti-γH2AX antibody (JBW301) (EMD Millipore), followed by AlexaFluor-488 conjugated goat-anti-mouse F(ab’)₂ fragments (Invitrogen, Thermo Fisher Scientific), and mounted using Vectashield™ antifade medium (Vectorlabs, Burlingame, CA). Multiple (>6) non-overlapping images were captured with a 40x objective lens on a Leica MD5500 B fluorescence microscope (Leica Microsystems, Wetzlar, Germany). A cutoff of >10 γH2AX foci per nucleus was applied as a marker of (induced) DNA damage. Cells with >10 γH2AX foci were counted manually from at least 6 non-overlapping images. For intranuclear γH2AX staining and DNA content analysis by flow-cytometry, cells were fixed and permeabilized using the Foxp3-staining buffer set (Affymetrix eBioscience) as per manufacturer’s instructions. Cells were stained with AlexaFluor-647 conjugated anti-γH2AX antibody (2F3: Biolegend, San Diego, CA) and propidium iodide (PI) (Invitrogen, Thermo Fisher Scientific).