Naive B Cell Isolation Kit II, human

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	. 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.	On day 7, B cells were harvested and cocultured with DC-T cells.

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.
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On day 7, B cells were harvested and cocultured with DC-T cells.

Publication protocol

Detection Immunogenetic of NitraTh-Based Vaccines by In Vitro Immunization System:The DCs were induced as above and stimulated with different NitraTh-based vaccines (HER2, HER2-Th, HER2-NitraTh, CB1, CB1-Th, or CB1-NitraTh). Naïve CD4+ T cells sorted from the same donor were cocultured with these DCs in the AIM-V medium (Gibco, USA) containing 0.5 ng/mL IL-12 and 1 ng/mL TGF-β (R&D Systems, USA) for 7 days. Culture medium and cytokines were renewed every 3 days. 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. On day 7, B cells were harvested and cocultured with DC-T cells. At 12 days later, the culture supernatants and cells were collected, and autoantigen-specific antibodies and antibody-secreting B cells were measured by ELISA and ELISpot assay, respectively.

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