EasySep™ Human Naïve B Cell Enrichment Kit

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	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).

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).

Publication protocol

Patient’s and biological parents’ blood/tissue samples were obtained after written informed consent through protocols established by the Pediatric Oncology Branch and the Lymphocyte Molecular Genetics Unit and approved by the Institutional Review Boards (IRB) of the National Institutes of Health (NIH). The parents separately provided consent for a single-patient clinical protocol that was approved by the University of Kentucky IRB. Anonymized buffy coat samples from healthy normal donors were provided by the NIH Blood Bank. Jurkat T cell (E6 and JPM50.6) and BJAB B cell lines were maintained in complete RPMI medium (RPMI 1640 (Lonza) supplemented with 10 % fetal bovine serum (FBS), 2 mM glutamine, and 100 U/ml each of penicillin and streptomycin (Invitrogen)). Peripheral blood mononuclear cells (PBMC) were isolated from whole blood or buffy coats using Ficoll-Hypaque-PLUS (Roche) density gradient centrifugation. Following erythrocyte lysis in ACK buffer (Lonza), pan T cells or naïve B cells were purified by negative selection using magnetic bead sorting kits (EasySep Human T cell Enrichment Kit, EasySep Human Naïve B cell Enrichment Kit, Stem Cell Technologies). 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). For ELISA and plasmablast assays, naïve B cells were stimulated with either anti-IgM F(ab’)2 (Jackson Immunoresearch, 10 μg/ml) or dextran-coupled anti-IgD or anti-IgM (Fina Biosolutions, 100 ng/ml) plus 1 μg/ml anti-CD40 (R&D Systems) and 10 ng recombinant human IL-21 (BioLegend). T cells were stimulated with 2 μg/ml each of anti-CD3ε and anti-CD28 mAbs (BD Biosciences) or MACS iBead particles (1:1 bead:cell ratio) loaded with biotinylated anti-CD2, anti-CD3ε, and anti-CD28 Abs (Miltenyi Biotec) in complete RPMI.

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