Easysep human b cell isolation kit

Human PBMC were obtained by density gradient centrifugation and human B-cells were negatively sorted from PBMC using EasySep Human B-cell Isolation Kit (StemCell) according to the manufacturer’s instructions. Purified B-lymphocytes were seeded at 1 × 10⁶ cells/ml in IMDM medium (Lonza) supplemented with 20% fetal bovine serum (Deutscher), 1% penicillin/streptomycin (Gibco) and stimulated for 4 days with 100 ng/ml human recombinant CD40L (Enzo Life Sciences) alone or with 50 ng/ml recombinant human IL-4 (Peprotech) or 2 μg/ml Gardiquimod (InvivoGen) or 2.5 μg/ml CpG oligodeoxynucleotide 2006 (InvivoGen) or 100 ng/ml IL-21 (R&D Systems) or 100ng/ml INFγ (R&D Systems) or 50 ng/ml Pam3CSK4 (InvivoGen) or 0.5 μg/ml goat anti-human kappa (Southern Biotech).

The study was approved in accordance with the University of Hawaii Institutional Review Board. B-NHL cell lines (Ramos and CA46) were obtained from American Type Culture Collection (ATCC, Manassa, VA) and cultured with RPMI 1640 medium and fetal calf serum. The cells were washed and re-suspended in 0.9% saline solution. Normal B-cells were isolated from peripheral blood using a negative selection Robosep kit (EasySep Human B-Cell Isolation Kit, Stemcell Technologies, Cambridge, MA) and re-suspended in saline solution as noted above.

B cell isolation was performed using the the EasySep Human B Cell Isolation Kit (StemCell Technologies). PBMCs were diluted in PBS supplemented with 2% FBS and 1 mM EDTA to a final concentration of 10⁷ cells/ml. Isolated B cells were resuspended in B-LCL-medium containing CpG ODN 2006 (2.5 μg/ml; InvivoGen) and holo-transferrin (30 μg/ml; Sigma-Aldrich). Previously quantified Epstein Barr virus–containing supernatant from B-95 cells was added to the cell suspension before plating 5 × 10⁵ cells per well in a round-bottom 96-well plate.

B cells were isolated from PBMCs by immunomagnetic negative selection using EasySep Human B Cell Isolation Kit (Stemcell Technologies) in accordance with the manufacturer’s instructions. B cells were cultured in StemMACS HCS Expansion Media XF (Miltenyi Biotec) supplemented with 1% streptomycin and penicillin (Invitrogen), 5% Human AB Serum (Valley Biomedical), and 125 IU/mL of IL4 (Miltenyi Biotec) at a density of 5 × 10⁵ cells/ml. B cells were expanded by crosslinking CD40 using Human CD40-Ligand Multimer Kit (Miltenyi Biotec) at a concentration of 8 U/ml in accordance with the manufacturer’s instructions. Media, IL4, and multimeric CD40L were refreshed every 3–4 days throughout the duration of all experiments.

PBMC samples were thawed in a 37°C water bath, and cells were immediately and gently added to pre-chilled thaw diluent (1:30 ratio) containing Plasma-Lyte A Injection, pH 7.4 (Baxter Healthcare Corp) with added heparin (10 IU/mL, StemCell Tech) and DNase I (0.01 mg/mL, Stem Cell Tech). Cells were centrifuged at 300x g for 5 min at room temperature and pellet resuspended in 1X RoboSep buffer (StemCell Tech) for subsequent B cell isolation. If cell aggregates were observed, cell suspensions were filtered with a 37 μm cell strainer (StemCell Tech). B cell isolation was performed using the EasySep Human B cell Isolation kit (StemCell Tech) with minor adjustments to the standard protocol, including performing isolation in a 96-well plate with proportionately scaled-down reagents. B cell purity was assessed via flow cytometry for select samples. Cells were stained with: CD19 PE (BD; clone HIB19), CD14 FITC (BD; clone M5E2), CD15 BV510 (BD; clone W6D3), CD3 BV421 (BD; clone SK7), CD56 PE-Cy7 (BD; clone B159), CD45 APC-Cy7 (BD; clone 2D1), and 7-AAD (BioLegend). Samples were acquired on a CytoFlex analyzer (Beckman Coulter), and data were analyzed using FlowJo v10 (FlowJo, LLC).