Human b cell nucleofector kit

MEC-1, EBV-B and human primary B cells were cultured in RPMI-1640 (Sigma-Aldrich, The Woodlands, TX) supplemented with 7.5% bovine calf serum at 37 °C in a humidified atmosphere with 5% CO₂.
Plasmids included murine Stat4 pRc/CMV (Addgene, Cambridge, MA), pcDNA3.1 (Invitrogen, Carlsbad, CA) and the pcDNA3-p66Shc constructs encoding human wild-type p66Shc or the respective p66ShcSA (S→A substitution at position 36) and p66ShcQQ (EE3→QQ substitutions at positions 132-133) mutants [4 (link)].
The esiRNAs used to silence STAT4 (EHU069141) in human cells, as well as unrelated control RLUC esiRNA (EHURLUC) were purchased from Sigma-Aldrich. p66shc silencing in human cells was obtained using the p66Shc-1 siRNA (GenScript Corporation, Piscataway, N.J).
Transfections were carried out using a modification of the DEAE-dextran procedure [38 (link)] or by electroporation. Assays were carried out after 24, 48 or 72 h.
Reconstitution of p66Shc expression in CLL B cells was obtained using the Human B-cell Nucleofector Kit (Amaxa Biosystems, Cologne, Germany) according to manufacturer's instructions as previously described [12 (link)].

Stable transfectants generated using the CLL-derived B-cell line MEC-1 and expressing human full-length p66Shc or the p66ShcQQ mutant were previously described (Stacchini et al., 1999 (link); Capitani et al., 2012 (link)). CLL cells were transiently transfected with 5 μg pcDNA3 or p66Shc-encoding pcDNA3 or p66ShcQQ pBabe vectors/sample using the Human B-cell Nucleofector Kit (Amaxa Biosystems, Cologne, Germany) as described (Patrussi et al., 2019 (link)). Assays were carried out after 24/48 h. For NF-AT translocation assays, Jurkat T cells were transiently transfected with the plasmid pEGFP/NFAT-1D (Plyte et al., 2001 (link)) by electroporation.

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.