MojoSort™ Human Naïve B Cell Isolation Kit

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	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
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.

Publication protocol

Peripheral blood mononuclear cells (PBMCs) collected from anonymous platelet donors were obtained from Gulf Coast Regional Laboratories (Houston, Texas). All human leukocyte packs were tested to be negative for human immunodeficiency virus, hepatitis B virus, hepatitis C virus, and human T-lymphocyte virus before shipment. For each experiment, blood packs were diluted with HBSS and overlaid on Ficoll-Paque Plus density gradient (GE Healthcare, Piscataway, New Jersey) and centrifuged at 1300× g for 25 min with low acceleration and brake. The PMBCs were isolated postcentrifugation, washed, counted and subjected to magnetic column-based isolation that enriched CD19+CD27- naïve human B cells (more than 95% purity). 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. The treated B cells were then activated by cocultured with sublethally irradiated CD40 ligand-L cells (1×104 cell/ml) in a 48-well cell culturing plate. Cells were cultured with recombinant human cytokines IL-2 (1 ng/ml), IL-6 (1 ng/ml) (Roche Applied Science, Indianapolis, Indiana), and IL-10 (4 ng/ml) (Biovision, Inc., Milpitas, California) for a total of 7 days. Soluble CD40L (100 ng/ml) (Enzo, Farmingdale, New York), IL21 (100 ng/ml), and IL2 (1 ng/ml) were added and cultured for 7 days with human B cells. B cells were activated with Pokeweed Mitogen (PWM) (15ug/ml) for 5 days. For LCK mRNA analysis, human B cells were treated with VH (0.02% DMSO) or TCDD (0.3, 3, and 30 nM) and cultured for a total of 3 days postactivation to verify the transcriptomic study (Kovalova et al., 2017). For all other studies, human purified naïve B cells were cultured for a total of 7 days. The LCK inhibitors were added on day 5 due to the upregulation of LCK protein expression. In the current study, responders were classified by a greater than 20% suppression of the IgM response in the presence of 10 nM TCDD when compared with VH control as measured by ELISA and ELISPOT.

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