Mouse monoclonal anti phospho histone h2ax antibody

LCLs were cultured 4 hours before fixation with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, Philadelphia, USA). Two hours before fixation, cells were counted and seeded into a poly-L-lysine-coated (Sigma-Aldrich) μCLEAR bottom 96-well plate (Greiner Bio-One) at a density of 75,000 cells per 100ul full media per well. LCL were then left for 2 hours in order to attach to the surface of the wells, fixed for 15 min at room temperature, permeabilized in 0.5% Triton X-100 in PBS for 20 minutes at 4°C and stained with primary and secondary antibodies and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) to visualize nuclei. To detect γ-H2AX we used mouse monoclonal anti-phospho-histone H2AX antibody (Millipore; #05-636). Alexa Fluor 488 from molecular probes (Invitrogen; #A-11034) was used, and fluorescent images were automatically taken for each well of the 96-well plate using an Opera High-Content Screening System (Perkin Elmer). Pictures were taken under non-saturating conditions using a 40x magnification lens to calculate the γ-H2AX nuclear signal intensity.

LCLs were cultured 4 hours before fixation with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, Philadelphia, USA). Two hours before fixation, cells were counted and seeded into a poly-L-lysine-coated (Sigma-Aldrich) μCLEAR bottom 96-well plate (Greiner Bio-One) at a density of 75,000 cells per 100ul full media per well. LCL were then left for 2 hours to attach to the surface of the wells, fixed for 15 min at room temperature, permeabilized in 0.5% Triton X-100 in PBS for 20 minutes at 4°C and stained with primary and secondary antibodies and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) to visualize nuclei. To detect γ-H2AX we used mouse monoclonal anti-phospho-histone H2AX antibody (Millipore; #05-636). Alexa Fluor 488 from molecular probes (Invitrogen; #A-11034) was used, and fluorescent images were automatically taken for each well of the 96-well plate using an Opera High-Content Screening System (Perkin Elmer). Pictures were taken under non-saturating conditions using a 40x magnification lens to calculate the γ-H2AX nuclear signal intensity.

After 1 h treatment with two doses of hydrogen peroxide, cells were trypsinized and seeded on a glass in a petri dish at different density. The slides were fixed with 4% paraformaldehyde (Sigma Aldrich, St. Louis, MO, USA), permeabilized in 0.2% Triton X-100 and blocked in PBS/BSA 1% for 30 min at RT. Slides were incubated with a mouse mono-clonal anti-phospho-histone H2AX antibody (Millipore) or an anti-53BP1 antibody (Novus Biologicals, Littleton, CO, USA) overnight at 4 °C, washed in PBS/BSA 1% and then exposed to the secondary Alexa 488-labeled donkey anti-mouse antibody (Invitrogen, Life Technologies, Carlsbad, CA, USA) for γH2AX and Alexa Fluor 488 labeled goat anti-rabbit (Invitrogen, Life Technologies, Carlsbad, CA, USA) for 53BP1, for 1 h at 37 °C. After washes in PBS/BSA 1% DNA were counterstained with DAPI (Sigma Aldrich) in Vectashield (Vector Laboratories, Burlingame, CA, USA). Cells were analyzed with fluorescence microscopy using an Axio Imager Z1 microscope (Carl Zeiss, Oberkochen, Germany) equipped with the Metacyte module of the Metafer automated capture software and a CCD camera (MetaSystems, Milano, Italy). The frequency of foci per cell were scored in 100 nuclei in at least two independent experiments.