Pathway-specific DSB repair analysis in HSPC and PBL was performed as described in Ref. (23 (link), 26 (link), 39 (link)). Briefly, actively cycling cells were transiently nucleofected with the DSB repair substrate HR-EGFP/5′EGFP (long homologies), detecting conservative HR, according to an Amaxa® protocol (Human B Cell Nucleofector Kit; Human CD34+ Cell Nucleofector Kit; Lonza, Cologne, Germany) via electroporation (Bio-Rad Laboratories, Hercules, CA, USA). While DSB formation within the substrate is usually induced by co-nucleofection of the I-SceI meganuclease expression plasmid pCMV-I-SceI, in the present study, the nucleofection mixture did not contain the expression plasmid. Instead, DSB were induced by exposing the cells 2–4 h after nucleofection to X-rays or heavy ions (carbon and calcium ions).
The assay monitors reconstitution of wild-type EGFP, so that EGFP-positive cells were quantified 24 h post-irradiation by the diagonal gating method in the FL1/FL2 dot plot (FACS Calibur® FACScan, Becton Dickinson, Heidelberg, Germany), as described in Ref. (40 (link)). All nucleofections were performed in triplicates. The transfection controls additionally contained pBS filler plasmid (pBlueScriptII KS, Stratagene, Heidelberg, Germany) and wild-type EGFP expression plasmid for normalization of repair frequencies.
The assay monitors reconstitution of wild-type EGFP, so that EGFP-positive cells were quantified 24 h post-irradiation by the diagonal gating method in the FL1/FL2 dot plot (FACS Calibur® FACScan, Becton Dickinson, Heidelberg, Germany), as described in Ref. (40 (link)). All nucleofections were performed in triplicates. The transfection controls additionally contained pBS filler plasmid (pBlueScriptII KS, Stratagene, Heidelberg, Germany) and wild-type EGFP expression plasmid for normalization of repair frequencies.
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