Purified naïve B cells were cultured in RPMI 1640 containing L-glutamine (Sigma Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (Sigma Aldrich), 10 mM HEPES (pH 7.4; Sigma-Aldrich), 0.1 mM nonessential amino-acid solution (Sigma- Aldrich), 1 mM sodium pyruvate and 40 μg/ml apo-transferrin (Sigma-Aldrich) and supplemented with 60 μg/ml penicillin and 100 μg/ml streptomycin. To induce class switch recombination, recombinant human CD40L (1μg/ml; R&D Systems, Minneapolis, MN, USA), Fab fragment anti-human IgM (Jackson Immunoresearch, West Grove, PA, USA), IL-2 (100 IU/ml; PeproTech) and IL-21 (50 ng/ml; PeproTech, Rocky Hill, NJ, USA) were added at the beginning of the culture. Cells were cultured in 96-well round bottom well plates (NuncTM, Roskilde, Denmark) for 5 days. Culture supernatants were collected for ELISA at the end of the culture.
Recombinant human cd40l
Manufactured by R&D Systems
Sourced in United States
Recombinant human CD40L is a cytokine that belongs to the tumor necrosis factor (TNF) superfamily. It is a type II transmembrane protein that can be cleaved into a soluble form. CD40L is primarily expressed on activated T cells and plays a crucial role in the activation and differentiation of B cells.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 2 (il 2), by Thermo Fisher Scientific (2 mentions)
Non essential amino acid solution, by Merck Group (2 mentions)
Fab fragment anti human igm, by Jackson ImmunoResearch (2 mentions)
Apo transferrin, by Merck Group (2 mentions)
Il 21, by Thermo Fisher Scientific (2 mentions)
Hepes, by Merck Group (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 8, by BioLegend (1 mentions)
Anti cd38 clone hb7, by BD (1 mentions)
Pe anti cd14 clone hcd14, by BioLegend (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
Annexin 5 fitc, by Immunotools (1 mentions)
Staphylococcal enterotoxin b from staphylococcus aureus seb, by Merck Group (1 mentions)
5 protocols using recombinant human cd40l
1
Naïve B Cell Class Switch Induction
2
Isolation and Activation of Monocytes
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RPMI 1640, fetal calf serum (FCS), penicillin and streptomycin were purchased from GIBCO. The Ficoll-Paque Plus used for cell separation was purchased from GE Healthcare (Munich, Germany). MACS CD14 cell isolation kit was obtained from Miltenyi Biotec. Bovine serum albumin was from Weiner Laboratorios (Santa Fe, Argentina). DMSO was purchased from Sigma-Aldrich (Dallas, USA). Annexin-V-FITC was obtained from ImmunoTools (Friesoythe, Germany). PE-conjugated mAbs anti- CD69 (clone FN50), anti-CD11b (clone D12), anti-CD49d (clone L25) and anti-CD38 (clone HB7), as well as FITC-anti-CD69 (clone FN50) and control Abs with irrelevant specificities (isotype control), monensin (BD GolgiStop™) and IL-8 ELISA Kit, were purchased from BD Biosciences (San Jose, USA). PC5-anti-CD19 (clone J3-119) was purchased from Beckman Coulter (Fullerton, USA). The following mAbs and recombinant human IL-8 were purchased from BioLegend (San Diego, USA): PE-anti-CD14 (clone HCD14), Alexa Fluor 488-anti-IL-8 (clone E8N1), FITC-anti-CXCR1 (clone 8F1) and PE-anti-CXCR2 (clone 5E8). Anti-human-IgM Ab was obtained from Jackson ImmunoResearch (West Grove, USA). Recombinant human CD40L was obtained from R&D Systems. Pam3CSK4 was purchased from Invivogen (San Diego, USA).
3
B Cell Cytokine Response to Stimuli
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3.5 × 105 MACS-purified B cells of patients with MS and healthy controls, as well as CD27-positive and CD27-negative B cells from healthy individuals, were plated in 96-well u-bottom plates and stimulated with either 4 µg/ml CpG or with 40 µg/ml affiniPure F(ab')2 fragment rabbit anti-human IgM (Fc5 Fragment Specific, Jackson Immuno Research) and 1 µg/ml recombinant human CD40L (R&Dsystem). Unstimulated samples served as controls. After 48 h of culture, supernatants were collected, and secreted IL-6 and IL-10 were determined by enzyme-linked immunosorbent assay (ELISA).
4
Naïve B Cell Class Switch Induction
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Purified naïve B cells were cultured in RPMI 1640 containing L-glutamine (Sigma Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (Sigma Aldrich), 10 mM HEPES (pH 7.4; Sigma-Aldrich), 0.1 mM nonessential amino-acid solution (Sigma- Aldrich), 1 mM sodium pyruvate and 40 μg/ml apo-transferrin (Sigma-Aldrich) and supplemented with 60 μg/ml penicillin and 100 μg/ml streptomycin. To induce class switch recombination, recombinant human CD40L (1μg/ml; R&D Systems, Minneapolis, MN, USA), Fab fragment anti-human IgM (Jackson Immunoresearch, West Grove, PA, USA), IL-2 (100 IU/ml; PeproTech) and IL-21 (50 ng/ml; PeproTech, Rocky Hill, NJ, USA) were added at the beginning of the culture. Cells were cultured in 96-well round bottom well plates (NuncTM, Roskilde, Denmark) for 5 days. Culture supernatants were collected for ELISA at the end of the culture.
5
Activation of Human B and T Cells
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An affiniPure F (ab ′ ) Fragment Goat Anti-Human IgA/IgG/IgM (H + L) (αBCR, 10 μg/ml) was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Anti-human CD40 monoclonal antibody (αCD40, 2 μg/ml), recombinant human CD40L (100 ng/ml) and recombinant human IFN-γ (20 ng/ml) were from R&D Systems (Minneapolis, MN, USA). Neutralizing anti-IFN-γ antibody (100 ng/ml) and anti-CXCR3 antibody (100 ng/ml) was from Biolegend (San Diego, CA, USA). We used Dynabeads Human T-Activator CD3/CD28 from Thermo Fisher Scientific (Waltham, MA, USA) for T cell activation with standard dose (25 μl/1 × 10 6 cells). Tofacitinib and Baricitinib were from Cosmo Bio (Tokyo, Japan). Staphylococcal enterotoxin B from Staphylococcus aureus (SEB, 1 μg/ml) and dimethyl sulfoxide (DMSO) was from Sigma-Aldrich (St. Louis, MO, USA). Anti-CD28/49d antibody (1 μg/ml) as a co-stimulator of SEB was from BD Biosciences (San Jose, CA, USA).
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