Quantifying DNA Double-Strand Breaks in PBMCs

The frequency of DNA DSBs within peripheral blood mononuclear cells (PBMC) was determined by microscopic quantification of focal accumulations of γ-H2AX and 53BP1^{19 (link)}, two marker proteins indicative for the presence of a DSB. For this purpose, PBMCs were isolated from blood samples by Ficoll Paque Plus (Merck, Darmstadt, Germany) density centrifugation. PBMCs were washed twice with PBS, fixed in 70% ethanol and stored at − 20 °C. Immunofluorescence staining and DSB foci analysis was performed as described previously^{19 (link),20 (link),42 (link)}. Using a Zeiss Axioimager Z2 epifluorescence microscope of the MetaSystems (Altlussheim, GER) ISIS imaging system equipped with appropriate single and dual-band filter sets co-localizing γ-H2AX + 53BP1 foci were counted in 100 well separated and morphologically intact cells by an experienced investigator (HS).

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Kaatsch H.L., Becker B.V., Schüle S., Ostheim P., Nestler K., Jakobi J., Schäfer B., Hantke T., Brockmann M.A., Abend M., Waldeck S., Port M., Scherthan H, & Ullmann R. (2021). Gene expression changes and DNA damage after ex vivo exposure of peripheral blood cells to various CT photon spectra. Scientific Reports, 11, 12060.

Publication 2021

Ficoll Paque Plus

53bp1 Blood Cells Centrifugation Ethanol Ficoll Immunofluorescence Microscope Peripheral blood mononuclear cells Proteins

Corresponding Organization : Bundeswehrzentralkrankenhaus Koblenz

Other organizations : Universität der Bundeswehr München, Johannes Gutenberg University Mainz

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Frequency of DNA DSBs within peripheral blood mononuclear cells (PBMC)

control variables

PBMC isolation using Ficoll Paque Plus density centrifugation
Washing of PBMCs with PBS
Fixation of PBMCs in 70% ethanol and storage at -20°C
Immunofluorescence staining and DSB foci analysis as described in previous studies

positive controls

None mentioned

negative controls

None mentioned

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