Human PBMC were isolated by density gradient separation on Lymphoprep (Axis-Shield PoC As, Norway), and cultured in the presence of Concanavalin A (0.625 μg/ml, Sigma-Aldrich) and LPS (5 μg/ml, Sigma-Aldrich) for 72 h. Cells were then cross-linked and lysed according to EpiTect ChIP OneDay kit (Qiagen). After sonication to shear the chromatin, cellular debris was removed by pelleting. After pre-clearing the chromatin, 1% of the sample was removed as “input fraction”. The rest of the sample was incubated with either 2 μg anti-Bcl-6 [85 (link), 86 (link)] (N3, Santa Cruz Biotechnology), or anti-Survivin [87 (link)] (10811, Santa Cruz Biotechnology). In each experiment, one sample with unspecific antibody was included as negative control for nonspecific binding. A known Bcl-6-targeted sequence within the first non-coding exon of the Bcl-6 gene was amplified and used as an internal positive control. The immune complexes were washed, the cross-links reversed and the DNA purified according to the EpiTect ChIP OneDay kit (Qiagen). The purified DNA was used as template in real-time amplification using different oligonucleotide pairs for p53 [52 (link)], Bcl-6 [86 (link)] and Blimp-1 [50 (link)]. PCR products were resolved on 2% agarose gels and visualized by ethidium bromide staining and quantified by the ChemiDoc equipment and Quantity One software (Bio-Rad Laboratories).
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