Dynabeads untouched human b cells kit

CD19⁺ B cells from healthy female subjects (n = 49) were isolated by negative selection using the Dynabeads Untouched Human B Cells Kit (Invitrogen). 1.5–3 × 10⁶ cells/ml ex vivo B cells were cultured in RPMI 1640 medium, supplemented with 20% FCS, 2mM l-glutamine and 100 U/mL penicillin/streptomycin. B cells from 32 of the 49 subjects were incubated with or without IFN-α 2b (1000 U/mL; PBL Assay Science) at 37 °C and 5% CO₂. Cells were harvested after 6 h or 20 h as indicated.
For immunostaining, human B Cell Isolation Kit II (Miltenyi Biotec). Ex vivo B cells (1 × 10⁶ cells/ml) were cultured in RPMI 1640 medium, supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine and 100 U/mL penicillin/streptomycin. B cells were stimulated with 10 µg/ml F(ab')2 Fragment Anti-Human IgG+IgM (Jackson ImmunoResearch) and either 0.1 µg/ml CD40L with 0.1 µg/ml Enhancer (Enzo) or 5 µg/ml resiquimod (Sigma). B cells were incubated with or without 10 nM bafilomycin A1 (Sigma) for 3 h before harvesting and with or without 1000 U/mL IFN-α 2b (PBL Assay Science) at 37 °C and 5% CO₂. Cells were harvested after 20 h or 27 h as indicated.

In order to analyse DSG1 and DSG3 specific B cells, B cells were isolated using Dynabeads Untouched Human B-cells kit (Life Technologies) according to the manufacturer's instruction. Then, purified B cells were incubated for 30 min at 4°C with histidine-tagged recombinant DSG1 or DSG3 (30 ng/μl). After washing, B cells were stained with anti-human IgG antibodies (BD Biosciences). Cells were then incubated with Fc Block (eBioscience), LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies), anti-human antibodies directed against CD19, CD27, IgM (BD Biosciences). Anti-histidine coupled with phycoerythrin (R&D Systems) was used to identify desmoglein-specific B cells.

Human B cells from healthy volunteers obtained via the French blood bank were separated from whole blood by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare) and Dynabeads Untouched Human B cells kit (Life Technologies) following the manufacturer’s instructions. Cell purity was assessed by flow cytometry analysis and was over 95% for B cells (CD19+). B cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (all reagents from Eurobio®) at 37 °C with 5% CO₂.

PBMC were prepared by Ficoll-Paque density centrifugation. Isolated B cells were prepared from PBMC using the Dynabeads Untouched Human B Cells Kit (11351D, Thermo Fisher Scientific) according to the manufacturer’s recommended protocol. Purity of the B cell suspension was assessed by flow cytometry, indicating a 99% CD19⁺ cell population consistent with high purity of B cells. B cells were resuspended 0.5 million/ml ml in RPMI 1640 containing 10% heat-inactivated human serum, 2 mM l-glutamine, 1 mM Na-pyruvate, 0.1 mM nonessential amino acids, 50 μM β-mercaptoethanol, streptomycin (100 μg/ml), and penicillin (100 U/ml), alone, or supplemented with IL-4 (20 ng/ml; PeproTech, Rocky Hill, NJ) plus anti-human IgA + IgG + IgM (30 μg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA) plus sCD40L (1000 ng/ml; Enzo Life Sciences, Farmingdale, NY). B cells (0.1 million) were added in 0.2 ml per well in 96-well U-bottom tissue culture plates and incubated at 37°C in 5% CO₂. After 24 hours, supernatants were harvested and frozen until evaluation.

Calcium ionophore A23187, ATP-agarose, γ-globulin, iron standard for AAS (1000 mg/l), phosphatidylcholine (PC), and penicillin/streptomycin were purchased from Sigma Aldrich (Taufkirchen, Germany).
Arachidonic acid was purchased from Biomol (Hamburg, Germany). The monoclonal mouse anti-5-LO antibody 6a12 was produced in-house. Fetal calf serum (FCS) was purchased from Biochrom AG (Berlin, Germany), SYPRO^® orange, Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640, the CloneJET PCR Cloning kit, Taq DNA polymerase, RNA-to-cDNA Kit, the Dynabeads Untouched Human B Cells kit, TRIzol Reagent and Turbo DNase were from Thermo Fisher Scientific (Waltham, MA, USA). The Nucleospin Gel and PCR Clean-up kit was from Macherey-Nagel, Düren, Germany. The EasySep™ Human T Cell Enrichment kit was purchased at Stemcell Technologies (Vancouver, Canada).
Oligonucleotides were synthesized by Eurofins MWG Operon (Ebersberg, Germany) and DNA sequencing was performed by Scientific Research and Development GmbH (Oberursel, Germany). The pCMV6-XL5 –B2M plasmid was bought from OriGene (Rockville, MD, USA), the plasmid pT3-5LO was a kind gift of Dr. Olof Rådmark (Karolinska Institutet, Stockholm, Sweden).

PBMC were isolated via a standard Ficoll gradient. Informed consent in writing was obtained from each donor. B cells were enriched from PBMC using Dynabeads Untouched Human B cells Kit (Thermo Fisher). For further analysis, B cells were incubated with human Fc block (BD Biosciences) for 10’ at RT, followed by biotinylated HBsAg for 30’ on ice (HBsAg by Roche Diagnostics; EZ-Link Sulfo-NHS-Biotinylation kit, Thermo Fisher). Memory B cell staining: 30’ on ice in PBS+0.5% BSA with live-dead stain (Thermo Fisher), anti-CD19-eF450 (eBioscience), anti-IgG-Pe-Cy7 (BD) and Streptavidin-APC (eBioscience). Cells were analyzed via flow cytometry on Cytoflex S (Beckman Coulter) or sorted with MoFlo II (Beckman Coulter) into lysis buffer, i.e. PBS+12U RNasin (Promega), 100 mM DTT (Thermo Fisher). PCR plates were sealed and stored at -80°C. IgG gene identification via PCR and sequencing from single B cells was performed as described elsewhere (16 (link)).