Peripheral blood mononuclear cells (PBMCs) were obtained using Ficoll-Hypaque density gradient centrifugation. CD4+ and CD8+ T cells were purified by negative selection (Miltenyi Biotec) and stimulated with human CD3/CD28/CD2 antibody-coated beads (Miltenyi Biotec) at 1:1 bead-to-cell ratio, in the presence of 50 IU/ml of rhIL-2 (Miltenyi Biotec) for three days. CD19+ B cells were purified by positive selection (Miltenyi Biotec) and stimulated with 8 IU/ml of CD40L (Miltenyi Biotec) in the presence of 50 IU/ml of IL-4 (Miltenyi Biotec) for three days. CD14+ monocytes were purified by positive selection (Miltenyi Biotec) and stimulated with 100 ng/ml of LPS (eBioscience) and 500 ng/ml of R848 (InvivoGen) for three days. Primary cells were maintained in RPMI 1640 medium (Corning) supplemented with 10% FBS, 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 2 mM of L-glutamine at 37°C/5% CO2.
Cd40l
Manufactured by Miltenyi Biotec
Sourced in United Kingdom
CD40L is a recombinant protein that functions as a ligand for the CD40 receptor. It is used in cell biology research applications.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by Miltenyi Biotec (3 mentions)
Rpmi 1640 medium, by Corning (2 mentions)
Ionomycin, by Fujifilm (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
Rhil 2, by Miltenyi Biotec (1 mentions)
24 well plate, by BD (1 mentions)
Anti igm antibody, by Jackson ImmunoResearch (1 mentions)
Cpg odn 2006, by InvivoGen (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate, by Fujifilm (1 mentions)
Golgiplug, by BD (1 mentions)
Anti cd19 fitc, by BD (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Fujifilm (1 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (1 mentions)
Entospletinib, by Selleck Chemicals (1 mentions)
6 protocols using cd40l
1
Isolation and Stimulation of Primary Immune Cells
2
CLL B-Cells Response to Apoptosis Induction
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MSCs were isolated from healthy donor bone marrow, as described by Naudot et al. [29 (link)]. MSCs were seeded at 2 × 104 cells/ml in 24-well plates (Falcon) in alpha minimum essential medium (MEM) supplemented with 10% FBS, penicillin/streptomycin (1%), L-glutamine (1%) and 0.5 ng/ml basic fibroblast growth factor (bFGF) and incubated overnight to allow cells adhesion. Freshly isolated CLL B-cells were cultured alone or on MSCs at 4x105cell/ml (ratio: 20:1) in RPMI medium. CLL cells were co-cultured with MSCs for 6 h (n = 12) or 24 h (n = 6) prior to AA treatment (250 μM). After 24 h, CLL cells were carefully removed and cell viability was assessed as described above.
CLL B-cells were stimulated with CpG-ODN2006 (1.5 μg/ml) (Invivogen), CD40L (50 ng/ml) + IL-4 (50 ng/ml) (Miltenyi) or anti-IgM antibody (10 μg/ml) (Jackson ImmunoResearch) (n = 7) or cultured in the presence of a combination of cytokines (as described in [30 (link)]) (n = 6) or in presence of 10% of the autologous patient’ serum (n = 10) and treated with AA for 24 h before cell viability/apoptosis was assessed in an annexin V-APC/7-AAD flow cytometry assay.
CLL B-cells were stimulated with CpG-ODN2006 (1.5 μg/ml) (Invivogen), CD40L (50 ng/ml) + IL-4 (50 ng/ml) (Miltenyi) or anti-IgM antibody (10 μg/ml) (Jackson ImmunoResearch) (n = 7) or cultured in the presence of a combination of cytokines (as described in [30 (link)]) (n = 6) or in presence of 10% of the autologous patient’ serum (n = 10) and treated with AA for 24 h before cell viability/apoptosis was assessed in an annexin V-APC/7-AAD flow cytometry assay.
3
CFSE Proliferation Assay for CD4+ T Cells
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Cell proliferation was determined using CFSE dilution assays. CD4+ T cells were positively selected with anti-CD4 microbeads. For staining with CFSE (Invitrogen, Carlsbad, CA), CD4+ T cells at 1 × 106 cells/ml in phosphate-buffered saline (PBS) were incubated with 5 μM CFSE for 10 min at 37 °C. Staining was terminated by adding RPMI 1640 containing 10% FCS at 4 °C, followed by one wash with PBS. CFSE-labeled CD4+ T cells were then seeded in 96-well plates (3.5 × 105 cells/well; Nunc A/S, Roskilde, Denmark), and either CD19+CD24hiCD27+ B cells or other B cells were added at a ratio of 1:1. CpG (ODN M362, 10 μg/mL; Enzo Life Sciences), CD40L (1 μg/mL; Miltenyi Biotec), phorbol 12-myristate 13-acetate (50 ng/mL; Wako), and ionomycin (1 μg/mL; Wako) were also added to each plate. CD4+ T cells were profiled by CFSE labeling after 5 days of incubation. Unstained cells were included in all experiments and were used to set the compensations on the flow cytometer.
4
Stimulation of Human B Cells
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PBMCs were resuspended (2 × 106 cells/mL) in medium and cultured in the presence of CpG (ODN M362, 10 μg/mL; Enzo Life Sciences, Farmingdale, NY) and CD40L (1 μg/mL; Miltenyi Biotec) for 24 h in 48-well flat-bottom plates. Nineteen h after stimulation, phorbol 12-myristate 13-acetate (PMA; 50 ng/mL; Wako, Osaka, Japan), ionomycin (1 μg/mL; Wako), and Golgiplug (Becton Dickinson, Franklin Lakes, NJ) were added to the culture. Twenty-four h after stimulation, cells were harvested and stained with anti-CD19-FITC, anti-CD24-PerCP Cy5.5, and anti-CD27-PE mAbs, then fixed and permeabilized with BD Cytofix/Cytoperm solution (BD Biosciences), and stained with anti-IL-10 mAb.
5
Activation of Naïve B Cells by BTLA
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PBMCs or sorted naïve B cells were rested for 30 min at 37°C in medium (RPMI 1640/10% FBS/1%Pen/Strep, all ThermoFisher). 96-well cell culture plates were coated for 2 hours at 37°C with 10 µg/ml anti-BTLA antibody (clone MIH26, Biolegend, San Diego, USA) or 10 µg/ml isotype control (mouse IgG2aκ, Biolegend) and washed two times with PBS. 1x106 PBMCs or 3-5x104 naïve B cells were seeded per well and pre-incubated with coated antibodies for 30 min. Naïve B cells were stimulated with 0.02 µg/ml IL-2 (Miltenyi), 0.02 µg/ml IL-10 (Miltenyi), 0.5 µg/ml aBCR F(ab)2 IgM/IgA/IgG (aBCR, Jackson ImmunoResearch, Ely, UK), 2.5 µg/ml CpG ODN 2006 (Miltenyi) and 0.5 µg/ml previously crosslinked CD40L (Miltenyi) or medium as control at 37°C and 5% CO2 in a humidified incubator. PBMCs were cultured under the same conditions and with the same stimulation cocktail mentioned above or with CpG or CD40L alone. In some experiments, 10 µM SYK inhibitor entospletinib (GS-9973, SelleckChem, Munich, Germany) dissolved in DMSO or DMSO (Sigma Aldrich, St. Louis, MO, USA) alone as a control was added to the culture. After five days, cells were harvested and subjected to staining. Concentrations were used according to prior titration experiments.
6
Isolation and Differentiation of Murine Naive B Cells
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Whole spleens were mechanically disassociated through a 40 m filter followed by red blood cell lysis with ammonium-chloride-potassium lysing buffer. Naïve mature B cells were isolated from spleen cells by immunomagnetic negative selection with anti-CD43 (Miltenyi Biotec). B cells were cultured in RPMI-1640 medium (Corning) supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (Corning), 1% MEM non-essential amino acids (Gibco), 1% sodiumpyruvate (Corning), 1% L-glutamine (Gibco), and -mercaptoethanol (50 M). B cells were stimulated with 1 ug/ml CD40L (BD Pharmingen) and 25 ng/ml IL-4 (R&D Systems) or with 0.1 ug/ml CD40L, 10 ng/ml IL-4, and 5 ng/ml IL-5 (R&D Systems) to monitor B cell processes during B cell differentiation or to monitor differentiation with increased plasma cell (PC) frequency, respectively (Shi et al., 2015) . For isolation of in vitro generated PCs used for Seahorse Extracellular Flux Analyzer assays, cultures stimulated for 5 days with CD40L, IL-4, and IL-5 were enriched for live cells using a dead cell removal kit (Miltenyi Biotec) followed by CD138-APC (BD Pharmingen) staining. CD138 + PCs were enriched by positive immunomagnetic enrichment of APC (Miltenyi Biotec). For Caspase 3/7, Mitrotracker Green, and TMRE staining experiments, in vitro generated PCs were isolated by immunomagnetic positive selection with anti-CD138 (Miltenyi Biotec).
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