B cells were isolated using immunomagnetic separation (Miltenyi Biotec) to negative sort splenic CD43− B cells. Cells were labeled with CTV (ThermoFisher scientific) at a concentration of 20 × 106 cells/ml in PBS. B cells for LPS differentiation were plated at 0.5 × 106 cells/ml and provided an initial stimulation of LPS (20 µg/ml; Sigma #L2630), IL-2 (20 ng/ml; eBioscience #14-8021), and IL-5 (5 ng/ml; eBioscience #14-8051). Half doses of LPS and cytokines were given on every 24 h. CD40L-stimulated cells were plated at 0.2 × 106 cells/ml and given daily stimulation with CD40L (100 ng/ml; R&D Systems), IL-4 (10 ng/ml; R&D Systems), and IL-5 (5 ng/ml; R&D Systems).
Cd40l
Manufactured by R&D Systems
Sourced in United States, France, Germany, United Kingdom
CD40L is a member of the tumor necrosis factor (TNF) superfamily. It is a type II transmembrane protein that is expressed on the surface of activated T cells. CD40L interacts with its receptor, CD40, which is expressed on the surface of B cells, dendritic cells, and other antigen-presenting cells. This interaction plays a crucial role in the activation and differentiation of B cells, as well as in the regulation of T cell-dependent immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by R&D Systems (16 mentions)
Lipopolysaccharides (lps), by Merck Group (12 mentions)
Ionomycin, by Merck Group (9 mentions)
Phorbol myristate acetate (pma), by Merck Group (6 mentions)
Anti igm f ab 2, by Jackson ImmunoResearch (6 mentions)
Cpg odn 2006, by InvivoGen (4 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (4 mentions)
Sodium pyruvate, by Corning (4 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Interleukin 6 (il 6), by R&D Systems (4 mentions)
Brefeldin a, by BD (3 mentions)
Ficoll paque plus, by GE Healthcare (3 mentions)
Mem non essential amino acid, by Corning (3 mentions)
Streptomycin, by Corning (3 mentions)
Penicillin, by Corning (3 mentions)
L glutamine, by Corning (3 mentions)
Hepes, by Corning (3 mentions)
Mmp 9, by R&D Systems (3 mentions)
Tnf α, by R&D Systems (3 mentions)
66 protocols using cd40l
1
Isolation and Activation of Murine B Cells
2
Regulatory B-cell Induction by IL-35
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For in vitro T-and B-cell culture experiments, PBMCs were cultured for up to 6 days with or without P pratense or CpG ODN 2006 (1 mg/mL; InvivoGen, San Diego, Calif) and CD40 ligand (CD40L; 0.01 mg/mL; R&D Systems, Abingdon, United Kingdom) for up to 48 hours, respectively. To investigate the effect of IL-35 on the induction of regulatory B (Breg) cells, PBMCs were cultured with CD40L (0.01 mg/mL; R&D Systems) and CpG ODN 2006 (1 mg/mL; InvivoGen) or LPS (100 ng/mL; Sigma-Aldrich, Dorset, United Kingdom) in the presence or absence of rhIL-35 (100 ng/mL; Enzo Life Sciences, Exeter, United Kingdom) for 48 hours. Cells were washed with culture medium and stimulated with phorbol 12-myristate 13-acetate (50 ng/mL; Sigma-Aldrich) and ionomycin (1 mg/mL; Sigma-Aldrich) in the presence of monensin (20 mg/mL; BioLegend, London, United Kingdom) or Brefeldin A (1:10; BD Biosciences) for 5 hours prior to staining. For B-cell culture, cells were blocked with Fc blocking agent (Miltenyi Biotec, Woking, United Kingdom). Cells were immunostained with cell-surface and intracellular antibodies (see the Methods section in this article's Online Repository) and acquired on BD FACSCanto II and BD LSRFortessa (BD Biosciences).
3
Enriched B cell culture and analysis
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Enriched B cells from HD and patients with seropositive rRA (2 × 10 6 cells/ml) were cultured for 7 days in complete medium with 10 ng/ml IL-2 under two different conditions:
(1) 50 ng/ml IL-21 (Biolegend) plus 1 µg/ml CD40L (R&D Systems, Minneapolis, MN, USA) and (2) 2•5 µg/ml anti-IgM/IgG F(ab) 2 fraction plus 50 ng/ml IL-21 and 1 µg/ mL CD40L [33, 34] . Supernatants were collected and stored at -20°C until the total immunoglobulin and autoantibody measurements. Cells were stained with Live/Dead following the manufacturer's instructions. After a blocking step, cells were labeled with anti-human CD19, CD21, CD24, CD27, CD38 and IgM monoclonal antibodies and analyzed by flow cytometry. The results are presented as frequencies.
(1) 50 ng/ml IL-21 (Biolegend) plus 1 µg/ml CD40L (R&D Systems, Minneapolis, MN, USA) and (2) 2•5 µg/ml anti-IgM/IgG F(ab) 2 fraction plus 50 ng/ml IL-21 and 1 µg/ mL CD40L [33, 34] . Supernatants were collected and stored at -20°C until the total immunoglobulin and autoantibody measurements. Cells were stained with Live/Dead following the manufacturer's instructions. After a blocking step, cells were labeled with anti-human CD19, CD21, CD24, CD27, CD38 and IgM monoclonal antibodies and analyzed by flow cytometry. The results are presented as frequencies.
4
LPS and CD40L Stimulation of Splenic B Cells
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For LPS stimulation, CD43− splenic B cells were seeded at 5 × 105 cells/ml in B cell media containing LPS (20 μg/ml, Sigma Aldrich), IL-2 (20 ng/ml, eBioscience), and IL-5 (5 ng/ml, eBioscience). Each subsequent 24 h the culture was supplemented with a 0.5× dose of LPS, IL-2, and IL-5 for the duration of the time course30 (link). For CD40L stimulation, cells were seeded at 5 × 105 cells/ml in B cell media containing CD40L (200 ng/ml, R&D Systems), IL-5 (5 ng/ml) and IL-4 (20 ng/ml, R&D Systems). Each subsequent day the culture was supplemented with a 0.5× dose of CD40L, IL-5, and IL-4 for the duration of the time course. GSK343 was a gift of Mike McCabe13 (link). GSK343 was resuspended in DMSO and added to the culture at the indicated doses. P3X63Ag8.653 cells (ATCC, CRL-1580) were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS.
5
Modulation of CD40L-induced CD95 expression
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Example 12
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- a. To test the interference of CD40L with anti-CD40 antibody mediated effects on cells, Ramos cells were treated with CD40L alone or in combination with agonistic anti-CD40 antibodies. Ramos cells were seeded in 96-well plates in RPMI containing 10% FCS at a cell density of 1.25×106 cells/ml. Antibodies were added to the wells at a concentration of 10 μg/ml and the plate was incubated for 10 minutes at 37° C., 5% CO2, 95% humidity. CD40L (R&D Systems) was then added to some wells to a final concentration of 10 μg/ml and the plate was incubated over night at 37° C., 5% CO2, 95% humidity. Cells were washed with DPBS and stained with a FITC-labelled antibody against CD95 (Miltenyi Biotech).
FIG. 17 shows that CD95 induction by CP-870,893 is strongly increased by the addition of CD40L, while co-treatment of all tested anti-CD40 antibodies of the invention reduce the effect of CD40L. The data indicates, that agonistic anti-CD40 antibodies of the invention which bind the CD40L binding on CD40, prevent synergistic and additive effects by CD40L and therefore allow controlled and safe pharmacology.
- a. To test the interference of CD40L with anti-CD40 antibody mediated effects on cells, Ramos cells were treated with CD40L alone or in combination with agonistic anti-CD40 antibodies. Ramos cells were seeded in 96-well plates in RPMI containing 10% FCS at a cell density of 1.25×106 cells/ml. Antibodies were added to the wells at a concentration of 10 μg/ml and the plate was incubated for 10 minutes at 37° C., 5% CO2, 95% humidity. CD40L (R&D Systems) was then added to some wells to a final concentration of 10 μg/ml and the plate was incubated over night at 37° C., 5% CO2, 95% humidity. Cells were washed with DPBS and stained with a FITC-labelled antibody against CD95 (Miltenyi Biotech).
6
Splenic B Cell Isolation and Culture
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Splenocytes were prepared by lightly disrupting 70 μm cell filters of the spleen. Erythrocytes were lysed with RBC lysis buffer (Solarbio, China). The splenocytes were subjected to magnetic bead sorting, using CD19 antibody-biotin (Miltenyi Biotec, Germany) and Anti-biotin microbeads (Miltenyi Biotec, Germany) according to the manufacturer’s recommendations, to obtain purified splenic B cells. Cells were then cultured in 1,640 medium containing 10% FBS, anti-IgM (10 μgmL−1, Thermo, United States), CD40L (100 ng mL−1, RD, United States) and IL-4 (20 ng mL−1, RD, United States), 100 U/ml penicillin and streptomycin in 5% CO2.
7
Isolation and Stimulation of B10 Cells
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Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMC) were isolated from heparin-treated PB and SF by Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) gradient centrifugation. PBMCs and SFMCs were cultured in RPMI 1640 containing L-glutamine (Life Technologies, Paisley, UK) supplemented with 100 U/μg/ml penicillin/streptomycin (Life Technologies, Paisley, UK), and 10% fetal bovine serum in 48-well flat-bottom plates for 48 h at 37 °C in 5% CO2. According to previous study [12 (link)], combination of CpG and CD40L stimulation could generate the most of B10 cells in human. Therefore, the cultured cells were stimulated with 10 μg/ml CpG ODN2006 (Invivogen, San Diego, USA) and 1 μg/ml CD40L (R&D Systems, Minneapolis, USA), or with phosphate-buffered saline (PBS) as control. For the last 6 h, 50 ng/ml phorbol myristate acetate (PMA) and 1 μg/ml ionomycin (Sigma-Aldrich, USA) were added to the stimulated cells; Brefeldin A (BFA, eBioscience, San Diego, USA), Golgi transport blocker, was added to all wells.
8
B Cell Activation and Ibrutinib Effects
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B cells were cultured in 96-well plates (2.5 × 104 cells/well) containing RPMI + 10% FBS superior (Merck, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Pan Biotech, Germany). B cells were stimulated with IL-4 (1,000 IU/ml, CellGenix, Germany), CD40L (2 µg/ml) and hemagglutinin (423 ng/ml) (both R&D, Canada) and cultured for 6 days (37 °C, 5% CO2). In addition, some cells were treated with ibrutinib (10 µM, Pharmacyclics, USA). FACS analysis of surface markers (CD19, CD39, CD73) was performed before and after stimulation.
9
T-cell Activation and Cytokine Profiling
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ConA (type IV) was purchased from Sigma (St. Louis, MO, USA). Mouse T-cell enrichment columns and neutralizing antibodies against B7.1, B7.2, OX40L and CD40L were obtained from R&D Systems (Minneapolis, MN, USA). Purified antibodies against mouse CD3, CD28 and cytometric Bead Array for mouse IFN-γ were purchased from BD Biosciences. Fluorochrome-conjugated antibodies against mouse CD3, CD4, CD8, CD19, CD44, CD45.1, CD45.2, B220, NK1.1, TCRαβ and CXCR3 were obtained from eBioscience (San Diego, CA, USA). ALT Detection Kits were purchased from NanJing JianCheng Biochemical Institute (Nan Jing, Jiang Su, China).
10
Neurite Arbor Analysis of Dissected Ganglia
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Dissected paravertebral SCG and prevertebral coeliac and superior mesenteric ganglia (PVG) were freed of adherent connective tissue using tungsten needles and were trypsinized and plated at very low density (~200 neurons per dish/well) in poly‐ornithine and laminin‐coated 35 mm tissue culture dishes (Greiner, Gloucestershire, UK) or 4‐well dishes (Starlab, Milton Keynes, UK) in serum‐free Hams F14 medium (Davies et al., 1993 ) supplemented with 0.25% Albumax I (Invitrogen, Paisley, UK). Neurons were grown with NGF, Fc control protein, recombinant CD40‐Fc, CD40L (R&D Systems), and caspase inhibitor III (Boc‐D‐FMK) (Merck) at the concentrations indicated. Analysis of the size and complexity of neurite arbors was carried out in 35 mm dishes 24 hr after plating. The neurite arbors were labeled by incubating the neurons with the fluorescent vital dye calcein‐AM (1:1,000, Invitrogen, Paisley, UK) at the end of the experiment. Images of neurite arbors were acquired by fluorescence microscopy and analyzed to obtain the Sholl profiles (Gutierrez & Davies, 2007 ).
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