Splenic B cells were purified by negative selection using anti-CD43 magnetic beads (Miltenyi, Bergisch Gladbach, Germany) according to manufacturer’s protocol. Cells were stimulated at 1×106 cells/ml in 96-well plates with 50 μg/ml LPS (Sigma Aldrich, St. Louis, MO), 10 μg/ml polyclonal anti-IgM, F(ab′)2 fragments (Jackson ImmunoResearch, West Grove, PA), or 10 μg/ml anti-CD40 (HM40-3) (BioLegend) + 100 U/ml IL-4 (Peprotech, Rocky Hill, NJ). After 72 hours, expansion was measured by flow cytometry using AccuCount 5.27 μm Blank Particles (Spherotech, Lake Forest, IL) according to manufacturer’s protocol.
Anti cd40 hm40 3
Manufactured by BioLegend
Anti-CD40 (HM40-3) is a purified Armenian hamster monoclonal antibody that binds to the mouse CD40 antigen. CD40 is a member of the tumor necrosis factor receptor superfamily and is expressed on B cells, dendritic cells, macrophages, and other cell types. The antibody can be used for flow cytometry, immunohistochemistry, and other research applications.
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Lab products found in correlation
Anti igm fab2 fragments, by Jackson ImmunoResearch (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Anti cd43 magnetic beads, by Miltenyi Biotec (1 mentions)
Accucount 5.27 μm blank particles, by Spherotech (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
2 protocols using anti cd40 hm40 3
1
Splenic B cell expansion assay
2
Exosome Depletion and B Cell Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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All fetal bovine serum (FBS) used in exosome cultures was first centrifuged at 100,000xg for 2 hours to deplete bovine exosomes. Splenic B cells were isolated using B220 positive magnetic bead selection as per manufacturer protocol (Miltenyi Macs system) and cultured for 3 days in 6 well plates at 1×106 cells/mL in cRPMI 1640 [13] containing 1 µg/mL anti-CD40 (HM40-3) (Biolegend) and 10,000 U/mL IL-4 (gift from Bill Paul NIH). When indicated, 10 µg/ml of anti-TNP- IgE (IGELb4) [14] (link) was present. When larger amounts of exosomes were needed, B lymphoma line M12.4.5 (M12) [15] (link) was used. M12 cells (1×106cells/mL – total cell number 3–5×108) were cultured for 24 hrs in cRPMI 1640/10% FBS (exosome-free as described previously) containing anti-CD40, IL-4, and anti-TNP (IGELb4) or anti-DNP IgE (10 µg/mL) [14] (link), [16] (link).
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