Optimisation of Ex Vivo Peripheral Blood Mononuclear Cell Culture and DNA Double Strand Break Repair Kinetics

The assessment and modelling of DNA double-strand break damage and repair is widely investigated throughout the literature. This optimisation study investigated the requirement of cell proliferation prior to treatment with chemotherapeutic agents to damage DNA and the optimal window of analysis for DNA double-strand break repair measurements with γ-H2AX. Peripheral blood mononuclear cells were collected from healthy volunteers and incubated with phytohaemagglutinin at final concentrations of 0, 0.25, 0.5, 1, 2.5, 5 and 10 µg/mL for 0, 24, 48, 72 and 168 h at 37 °C, 5% CO2, and proliferation was measured via spectrometry (MTS assay). This study, detailed in this methodology paper, found that peripheral blood mononuclear cells must be proliferated prior to the chemical induction of DNA double-strand breaks. The window for assessment of early DNA double-strand break repair was determined to be one hour after removal of the DNA damaging agent.