Anti human igm igg

The blood was obtained from 6 healthy donors. CD19⁺B-cells were isolated from PBMCs using EasySep Human B Cell Enrichment Kit (STEMCELL Technologies, Vancouver, BC, Canada). Purified B-cells (1x10^5 cells) were maintained in 96-well cell culture plate in RPMI 1640 medium supplemented with 10% FBS (Invitrogen), 40 ng/mL IL-4 (PeproTech Inc., NJ, USA) and 10 μg/mL CD40L (PeproTech Inc., NJ, USA) in the presence or absence of anti-human IgM+IgG (eBioscience, San Diego, CA, USA). Total RNA was extracted from cell pellets using the RNAqueous-Micro Total RNA Isolation Kit (Thermo Fisher Scientific, MA, USA) and were incubated for 1, 6 and 24 hours. qRT-PCR reactions were performed in 384-well plates with TaqMan gene probes and primers designed by Life Technologies for TAGLN2 and three reference genes, RPS18 (assay ID: Hs01375212_g1), RPLP0 (assay ID: Hs00420895_gH) and YWHAZ (assay ID: Hs01122445_g1). These reactions were performed as described above. TAGLN2 mRNA expression was normalized to the mean of three reference genes using the 2-ΔΔCt method. Data are presented as fold change relative to expression levels of non-stimulated controls.

Peripheral blood from normal volunteers was obtained through an Institutional Review Board-approved protocol. Peripheral blood mononuclear cells (PBMCs) were first purified from blood by density gradient centrifugation using Ficoll-Paque. Human B cells were then purified from PBMCs by negative selection (eBioscience Magnisort Human B cell enrichment kit). B-cell purity was increased from 4% to >70% as measured by FACS analysis (FACSCalibur and CellQuest software; BD Biosciences) using anti-CD19 PE conjugated antibody (eBioscience). Purified B cells were seeded at a final concentration of 0.1 × 10⁶ cells/mL and cultured with 2 µg/mL human CD40L (eBioscience) + 5 µg/mL anti-human IgM/IgG (eBioscience) + 100 µg/mL hIL-2 (R&D Systems) + 100 µg/mL hIL-21 (R&D Systems). All B cells were cultured in RPMI 1640 supplemented with 10% (vol/vol) heat-inactivated FCS, 5 mM Hepes, 2 mM l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM 2-mercaptoethanol.