PBMCs were cultured at a concentration of 2×106 cells/ml in RPMI-1640 containing L-glutamine, 10% FCS and antibiotics in medium only or stimulated with 10 μg/ml CpG-B (InvivoGen, California, USA) and 1 μg/ml CD40L (InvivoGen) for 48 h. For the last 5 hours of the culture, an additional stimulation with 50 ng/ml PMA and 1 μg/ml Inomycin (Sigma-Aldrich, MO, USA) in the presence of Golgistop used at 1:1000 dilution (BD) was performed. Cells were first stained for cell surface markers PerCp anti-CD19, FITC anti-CD10 and V450 anti-CD27 in addition to LIVE/DEAD staining. In next step cells were fixed and permeabilized using BD Cytofix/Cytoperm kit and stained with PE anti-IL-6 (MQ2-6A3) and matching isotype control, both from (BD). Cells were washed and fixed in 1% formaldehyde before the analysis were conducted using a Cyan flow cytometer. Collected data were analysed using FlowJo, version 9.4.11 (Tree star).
Cd40l
Manufactured by InvivoGen
Sourced in United States
CD40L is a cell surface protein that belongs to the tumor necrosis factor (TNF) superfamily. It is primarily expressed on the surface of activated T cells and plays a crucial role in the activation and regulation of B cells.
Automatically generated - may contain errors
Lab products found in correlation
Cpg b, by InvivoGen (2 mentions)
Golgistop, by BD (2 mentions)
L glutamine, by Corning (2 mentions)
Sodium pyruvate, by Corning (2 mentions)
Roswell park memorial institute (rpmi), by Corning (2 mentions)
Hepes, by Corning (2 mentions)
Recombinant human il 3, by R&D Systems (2 mentions)
Mem non essential amino acid, by Corning (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Corning (2 mentions)
Penicillin, by Corning (2 mentions)
Imiquimod, by Thermo Fisher Scientific (2 mentions)
Cpg a, by InvivoGen (2 mentions)
Cd40l, by R&D Systems (2 mentions)
Inomycin, by Merck Group (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
4 protocols using cd40l
1
IL-6 Expression in CpG-B and CD40L Stimulated PBMCs
2
Purification and Stimulation of Plasmacytoid Dendritic Cells
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Prior to cell culture, pDCs were purified by sorting to be free of tDCs (AXL−), following the gating strategy in Figure S3A . 10,000 sorted bona fide pDCs were cultured in 200 μL R10 complete media consisting of RPMI (Corning) with 10% FBS, 2 mM L-glutamine (Corning), 100 IU Penicillin, 100 mg/mL Streptomycin (Corning), 25 mM HEPES (Corning), 1 mM Sodium Pyruvate (Corning), 100 mM MEM Nonessential Amino Acids (Corning) and 55 mM 2-Mercaptoethanol (GIBCO) in 96 well U-bottom plates at 37°C. All conditions included 10 ng/mL recombinant human IL-3 (R&D Systems; carrier-free) for pDC survival. Stimulation conditions were 100 ng/mL CD40L (R&D Systems; carrier-free) or 5 μg/mL Imiquimod (R837, Invitrogen). For ATAC-seq, 4–6 wells were plated per condition. After 2 days, cells were pooled and sorted as FSC-A, SSC-A, Live, Singlets, CD123+ HLA-DR+ (see Figure S4A for sorting strategy). For analysis of IFN-I production in Figure 6G , pDCs were re-sorted into “pDC-like,” “tDC-like,” and “cDC-like” after 2 days of stimulation with CD40L, then stimulated for 24h with 5 μg/mL CpG-A (ODN 2216, Invivogen).
3
Measuring IL-10 Production in PBMCs
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The Bregs show an immunosuppressive function by the production of IL-10. For measuring IL-10 production, we stimulated the thawed PBMCs of five PR and five HC in two ways [according to Siewe et al. (31 (link), 33 (link))]. First, the cells (106 PBMCs/ml) were incubated for 48 h with 10 µg/ml CPG-B (oligodeoxynucleotide-2006), 2 µg/ml PAM (palmitoyl-3-cysteine-serine-lysine-4), and 2 µg/ml CD40L (all InvivoGen, San Diego, CA, USA) at 37°C and in 5.0% CO2, and during the last 5 h, the cells were supplemented with 1 µg/ml ionomycin, 50 ng/ml phorbol-12-myristate-13-acetate (PMA), 50 µg/ml brefeldin A (all Sigma), and 1 µl/ml monensin (BD Golgi Stop™ BD Biosciences). Second, we only stimulated the cells with ionomycin/PMA. After 2 h of incubation at 37°C/5.0% CO2, brefeldin A and monensin were added, and the cells were incubated for another 5 h. After the incubation, the cells were washed and stained intracellularly for IL-10. The background range was <0.02% for IL-10 production.
4
Purification and Stimulation of Plasmacytoid Dendritic Cells
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