High‐throughput measurement of double strand break global repair phenotype in peripheral blood mononuclear cells after long‐term cryopreservation

Abstract

AbstractPeripheral blood mononuclear cells (PBMCs) are a useful model for biochemical assays, particularly for etiological studies. We describe here a method for measuring DNA repair capacity (DRC) in archival cryogenically preserved PBMCs. To model DRC, we measured γ‐H2AX repair kinetics in thawed PBMCs after irradiation with 3 Gy gamma rays. Time‐dependent fluorescently labeled γ‐H2AX levels were measured at five time points from 1 to 20 h, yielding an estimate of global DRC repair kinetics as well as a measure of unrepaired double strand breaks at 20 h. While γ‐H2AX levels are traditionally measured by either microscopy or flow‐cytometry, we developed a protocol for imaging flow cytometry (IFC) that combines the detailed information of microscopy with the statistical power of flow methods. The visual imaging component of the IFC allows for monitoring aspects such as cellular health and apoptosis as well as fluorescence localization of the γ‐H2AX signal, which ensures the power and significance of this technique. Application of a machine‐learning based image classification improved flow cytometry fluorescent measurements by identifying apoptotic cells unable to undergo DNA repair. We present here DRC repair parameters from 18 frozen archival PBMCs and 28 fresh blood samples collected from a demographically diverse cohort of women measured in a high‐throughput IFC format. This thaw method and assay can be used alone or in conjunction with other assays to measure etiological phenotypes in cryogenic biobanks of PBMCs.