Class b cpg oligonucleotide

Exponentially growing B95-8 cells were seeded at 1×10⁶ cells/mL and incubated for 3d at 37°C, 5% CO₂. B95-8 supernatant was harvested by centrifugation at 300×g and 4°C and passed through a 0.45µm filter. PBMCs were suspended at 1–5×10⁶ cells/ml in RPMI-1640 media with 20% FBS followed by an addition of 0.5–1 volumes of B95-8 supernatant with or without 10µg of a human TLR9 ligand (ODN 2006, Class B CpG oligonucleotide, Invivogen). Cells were incubated overnight at 37°C in the presence of 5% CO₂ and 0.5µg/ml Cyclosporine A, and an equal volume of media was added the next day. Cells were cultured until they formed macroscopically visible clusters and then maintained at concentrations of 0.5–1×10⁶ cells/ml. For ER stress induction, EBV-transformed cells were seeded at 1×10⁶ cells/ml and incubated with 100nM thapsigargin (Sigma) for six hours. Cells were harvested, washed in PBS and processed for RNA isolation as described below.

B cells were negatively selected using EasySep™Human B Cell Enrichment Kit (Stem cell technologies) and labeled with CFSE (final working solution 1μM) using the CellTrace™ CFSE Cell Proliferation Kit (Life Technologies). Primary B cells were stimulated with 10ug/ml anti-IgM mix (AffiniPure Goat Anti-Human IgA+IgG+IgM (H+L), Jackson Immunoresearch Lab) + 1μg/ml recombinant human Soluble CD-40 Ligand (CD40L, ProSpec) + 1μg/ml Class B CpG oligonucleotide (Invivogen) for 5 days. Data was analyzed for proliferation index with FlowJo (Version 9).