Cell Isolation Naive B cell

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Protocol tips
. On day 5, human naïve B cells (purity of >95%, viability of >90%) that were sorted by Human Naïve B Cell Isolation Kit II (Miltenyi, Germany) were activated with CpG ODN 2006 (InvivoGen, USA) and NitraTh-based vaccines mentioned above.
Downstream tips
On day 7, B cells were harvested and cocultured with DC-T cells.
Protocol tips
B cells were isolated from PBMCs using the EasySep™ Direct Human Naïve B Cell Isolation Kit (STEMCELL Technologies) according to the manufacturer's instructions. B cells were centrifuged (500 × g for 5 minutes), the supernatant was removed, protein was isolated, and western blotting performed as described previously.
Protocol tips
Total (CD19+) or naïve (CD19+, CD27+, IgM+) B cells were isolated from peripheral blood mononuclear cells (PBMC) of healthy blood donors using a negative selection kit (Human naïve B cell isolation kit, Human B cell isolation kit, StemcellTM Technologies, Grenoble, France) after density gradient centrifugation (Ficoll®Paque Plus, GE Healthcare Life sciences, Velizy-Villacoublay, France).
Downstream tips
The cell purity was then controlled by flow cytometry on a Cytomics FC500 System (Beckman-Coulter, Fullerton, CA) using CD19+ or CD19+/CD27− staining (Beckman Coulter, Villepinte, France).
Protocol tips
This negative selection was conducted using the MojoSort human naïve B cell isolation kit (Biolegend, San Diego, California) following the manufacturer’s instructions. Purified human B cells at the concentration of 1 ×106 cells/ml were then treated with either 0.02% DMSO (VH) or various concentration of TCDD.
Protocol tips
CD10+ immature/transitional B cells were subsequently depleted using magnetic beads (CD10+ Positive Selection beads, Stem Cell Technologies). Naïve B cell stimulations were performed in complete RPMI medium using Staphylococcus aureus Cowan I (SAC, Sigma) + 200U/ml rIL-2 (Peprotech), anti-IgM F(ab’)2 (Jackson Immunoresearch, 25 μg/ml) −/+anti-CD40 (1 μg/ml) or recombinant human BAFF (200 ng/ml) (R&D Systems).
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