Anti human cd40 tnfrsf5 antibody

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by density gradient centrifugation (Lymphoprep™; StemCell, Technologies Inc., Vancouver, BC, Canada). PBMCs were resuspended in culture medium: RPMI-1640 formulated with glutamine (300 mg/L) (Irvine Scientific, Santa Ana, CA, USA) and supplemented with 10% heat-inactivated fetal calf serum (Cytiva, Marlborough, MA, USA) and antibiotics (penicillin and streptomycin) (EuroClone, Milan, Italy).
PBMCs were cultured (1 × 10⁶ cell/mL) in 24-well flat bottom plates (for ROS and autophagy evaluation) or 96-well plates (for mitochondrial evaluation) and incubated in the absence (unstimulated) or presence of specific B-cell stimulus combinations: F(ab)2 goat anti-human IgA + IgG + IgM (anti-BCR) (5 µg/mL; Jackson ImmunoResearch, West Grove, PA, USA), anti-human CD40/TNFRSF5 antibody (1 µg/mL; R&D Systems, Minneapolis, MN, USA), CpG-oligodeoxinucleotide (ODN) (0.6 µg/mL; CpG-ODN 2006 type B; InvivoGen, San Diego, CA, USA), and human recombinant IL-21 (100 ng/mL; BioSource, Blue Bell, PA, USA). Additionally, in cultures aimed at autophagic flux evaluation, bafilomycin A1 (10 nM; Sigma-Aldrich, St. Louis, MO, USA) was added to inhibit autophagosome degradation. Cultures were maintained for 24 h at 37°C in a 5% CO₂ atmosphere.

Peripheral blood mononuclear cells (PBMCs) isolated from heparinized blood by density gradient centrifugation were re-suspended in RPMI-1640 medium supplemented with 10% heat inactivated fetal calf serum and antibiotics (penicillin-streptomycin). In all, 1–4 × 10⁶ cells were cultured in 24-well plates and stimulated with F(ab)₂ goat anti-human IgA+IgG+IgM (5 μg/mL; Jackson ImmunoResearch) or with anti-human CD40/TNFRSF5 antibody (1 μg/mL; R&D Systems) in the presence or absence of human recombinant IL-21 (100 ng/mL; Biosource). Cultures were maintained at 37 °C in a 5% CO₂ atmosphere during 20 h.