Affiliations: [a] BTU Cottbus-Senftenberg, Institute of Biotechnology, Senftenberg, Germany
| [b] Department of Hematology, Oncology and Palliative Care, Brandenburg Medical School, University Hospital Brandenburg, Germany
Corresponding author: Stefan Rödiger, BTU Cottbus-Senftenberg, Institute of Biotechnology, Universitätsplatz 1, 01968 Senftenberg, Germany. Tel.: +49 3573 85936; E-mail: [email protected].
Abstract: Chemotherapy and radiation therapy are used in malignant oncological diseases to increase the level of DNA double strand breaks (DSBs) in tumor cells. Unrepaired DSBs may either kill a cell or induce terminal arrest. Those DNA damages can be detected by fluorescence imaging of phosphorylated histone protein H2AX (γH2AX). Peripheral blood mononuclear cells (PBMCs) are easily available human material, which are usually stored by cryopreservation for repeated experiments or postponed analysis. As DNA DSBs can be introduced through several different stressors, it was of interest to investigate the potential impact of cryopreservation, on the formation of DSBs in human PBMCs. The PBMCs were cryopreserved in four different media, containing different proportions of Dulbecco’s Modified Eagle Medium (DMEM), fetal calf serum (FCS) and dimethyl sulfoxide (DMSO) as taken from the literature. Immunofluorescence staining of γH2AX was performed on freshly isolated and cryopreserved PBMCs. A significant reduction on relative γH2AX level, after treatment with 100 μM etoposide (ETP), was observed for cryopreserved cells in medium containing DMEM, which seemed to be the limiting factor on DNA DSB formation. No change was observed for untreated PBMCs. Therefore, DSBs of cryopreserved human PBMCs could be used to investigate the influence of chemotherapeutic drugs or radiation therapy, assuming that the cryopreservation follows a standardized protocol with a tight control of potential DSB inducing stressors.
Keywords: DNA double strand breaks, γH2AX, cryopreservation, human PBMCs, automated microscopy