4.6. Immunofluorescence

Then, 50,000 D341 treated cells resuspended in PBS were spun in Thermo Scientific Cytospin 4 (Thermo Fisher Scientific, Waltham, MA, USA) at 500 rpm, for 10 min, and then processed for immunofluorescence. All cell-lines cells were either fixed for 10 min at RT with PFA 4% or, for γH2AX, 53BP1 and RAD51 staining, fixed 5 minutes at RT, using PFA 2%, treated for 10 minutes at RT using CSK buffer (100 mmol/L of NaCl, 300 mmol/L of sucrose, 3 mmol/L of MgCl2, 10 mmol/L of PIPES (pH 6.8) and 0.7% Triton), and fixed again, for 5 minutes, at RT, using PFA 2%. Subsequently, cells were permeabilized with 0.1% Triton X-100, in PBS, for 10 min, saturated in 5% BSA in PBS for 30 min, and incubated with a primary antibody for 2 h, at RT. Primary antibodies were detected with anti-rabbit Alexa Fluor 488 or 555 (Molecular Probes, Invitrogen), anti-mouse Alexa Fluor 488 or 555 (Molecular Probes, Invitrogen), used at 1:1000 dilution, for 30 min. Cells were counterstained with 0.5 mg/mL of DAPI for 10 min and washed with PBS. Finally, cell slides were mounted with Prolong (Thermo Fisher Scientific). TUNEL assay was performed 72 h after transfection in D283 and D341, using the TMR red In Situ Cell Death Detection Kit (Roche, Basel, Switzerland), according to the manufacturer’s protocol.