PBMC were prepared by Ficoll-Paque density centrifugation. Isolated B cells were prepared from PBMC using the Dynabeads Untouched Human B Cells Kit (11351D, Thermo Fisher Scientific) according to the manufacturer’s recommended protocol. Purity of the B cell suspension was assessed by flow cytometry, indicating a 99% CD19+ cell population consistent with high purity of B cells. B cells were resuspended 0.5 million/ml ml in RPMI 1640 containing 10% heat-inactivated human serum, 2 mM l -glutamine, 1 mM Na-pyruvate, 0.1 mM nonessential amino acids, 50 μM β-mercaptoethanol, streptomycin (100 μg/ml), and penicillin (100 U/ml), alone, or supplemented with IL-4 (20 ng/ml; PeproTech, Rocky Hill, NJ) plus anti-human IgA + IgG + IgM (30 μg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA) plus sCD40L (1000 ng/ml; Enzo Life Sciences, Farmingdale, NY). B cells (0.1 million) were added in 0.2 ml per well in 96-well U-bottom tissue culture plates and incubated at 37°C in 5% CO2. After 24 hours, supernatants were harvested and frozen until evaluation.
Scd40l
Manufactured by Enzo Life Sciences
Sourced in Germany
SCD40L is a lab equipment product offered by Enzo Life Sciences. It is a lab-scale instrument designed for specific applications. The core function of SCD40L is to perform a particular laboratory procedure, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Anti human igm igg iga, by Jackson ImmunoResearch (1 mentions)
Dynabeads untouched human b cells kit, by Thermo Fisher Scientific (1 mentions)
Vitd3, by Merck Group (1 mentions)
Cd14 specific immunobeads, by Miltenyi Biotec (1 mentions)
Rhil 10, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Megacd40l, by Enzo Life Sciences (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by GE Healthcare (1 mentions)
L glutamate, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Il 10, by R&D Systems (1 mentions)
Streptavidin, by Roche (1 mentions)
Tapi 0, by Bio-Techne (1 mentions)
Cellstar, by Greiner (1 mentions)
5 protocols using scd40l
1
Isolation and Activation of Human B Cells
2
Generation and Characterization of Dendritic Cells
3
Knocking Out CD40 in Human Kidney Cells
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Human proximal tubule epithelial cells (HK2; CRL‐2190; ATCC, Manassas, VA) were transfected with a CRISPR (clusters of regularly interspaced short palindromic repeat)/Cas9 (CRISPR‐associated protein‐9) vector (sc‐40024; Santa Cruz Biotechnology, Santa Cruz, CA) to knock out the human Cd40 gene. Vector‐expression cells were sorted by monitoring GFP (green fluorescent protein) signaling using flow cytometry. Up to 3000 GFP‐positive cells were cultured and expanded to certain confluency, and CD40 expression was examined with western blot and reverse‐transcription PCR. Both wild‐type HK2 cells and CD40‐knockout cells (HK2‐CD40KO) were treated with/without sCD40L (100 ng/mL; Cat. # ALX‐522‐110‐C010; Enzo Life Sciences Inc, Ann Arbor, MI) for 24 hours at a concentration we have previously shown to stimulate CD40 signaling.23 In addition, cells were treated with TNF‐α (tumor necrosis factor alpha; 10 ng/mL; Cat. # 300‐01A; PeproTech Inc, Rocky Hill, NJ) for 24 hours as a positive control for triggering inflammatory responses.24 After 24 hours, cells were washed with 2 mL of PBS twice, and cell lysate was collected for testing gene expression.
4
CLL Cell Stimulation and Cytospin Preparation
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Two million viable primary CLL cells per milliliter were grown in RPMI medium supplemented with 10% FBS (Gibco), 1% PEST (Gibco), and 1% l -glutamate (Gibco), supplemented with 25% vol/vol serum-free supernatant from the T cell hybridoma cell line MP6 (Rosén et al., 1986 (link)), as a source of thioredoxin (Söderberg et al., 1999 (link); Nilsson et al., 2000 (link)). For stimulation of the BcR, the modified RPMI medium was supplemented with 3 µg/ml AffiniPure F(ab′)2 fragment rabbit anti–human IgM (Jackson ImmunoResearch Laboratories, Inc.), 10 ng/ml IL-2 (GE Healthcare), and 1 µg/ml streptavidin (Roche). To stimulate the CD40 signaling pathway, cells were treated with the modified RPMI medium supplemented with 100 ng/ml sCD40L (Enzo Life Sciences), 25 ng/ml IL-4 (R&D Systems), and 100 ng/ml IL-10 (R&D Systems). Cells were stimulated for 15 min at 37°C (5% CO2). Cytospins were prepared using 150,000 cells per slide and a Cellspin I cytocentrifuge (Tharmac) at 500 rpm for 2 min.
5
HUVEC Isolation and CD40-Mediated Signaling
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Human umbilical vein endothelial cells (HUVEC) were isolated from freshly obtained umbilical cords provided by local hospitals with permission of the Ethics Committee (S-383/2013). Cell culture plates CellStar™ were obtained from Greiner Bio-One, Germany. Cell culture media and supplements were purchased from PromoCell, Germany. Cultivation was carried out as previously described [10 (link)]. Only cells carrying the CC genotype of the CD40 gene (rs1883832) in passage one were used for all experiments. HUVEC were stimulated with 200 ng/mL sCD40L (Enzo Life Science, Lörrach, Germany) or 100 U/mL TNFα (Biomol, Hamburg, Germany). Messenger RNA and protein levels were examined after 8 and 24 h, respectively. Soluble CD40 protein levels were determined in the cell culture supernatant after 24 h. Expression of MCP-1 was examined after 12 h of incubation with TAPI-0 following another 8-h or 12-h stimulation for mRNA or protein detection, respectively. For ADAM17 inhibition, the medium was supplemented with 30 µmol/L TAPI-0 (Tocris, Wiesbaden, Germany) dissolved in DMSO 1 h before the stimulation started. The final concentration of DMSO in the medium was 0.1% (v/v), and the controls received DMSO only.
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