Primary B cells were negatively isolated from healthy donor PBMCs (n = 4) using the B-cell isolation kit (Miltenyi Biotech) and cultured in RPMI-1640 (Gibco), containing 2 mM L-glutamine, 20% fetal bovine serum (FBS; Gibco), 100 μg/ml streptomycin and 100 IU/ml penicillin (Sigma-Aldrich), at 37°C in 5% CO2. Lymphoma cells from six MCL patients (median age 58, range 50–76 years; all male) affected by MCL in leukemic phase (>90% CD19+ cells) were also used. Written informed consent was obtained from all patients and donors. The study was reviewed and approved by the Internal Review Board (IRB) of the Centro di Riferimento Oncologico CRO (IRCCS) of Aviano (IRB-03-2010). Cells were activated with soluble CD40L (Alexis, 1 μg/ml), 1 μg/ml of the corresponding enhancer (Enzo Life Sciences), 10 ng/ml of IL-4 (R&D Systems), and 2 μg/ml F(ab’)2 fragment Goat Anti-Human IgM (Jackson ImmunoResearch), and treated for 72 or 96 hours with 30 ng/ml Pam3CSK4 (TLR1/2 ligand; Imgenex), or 75 ng/ml purified flagellin (TLR5 ligand; Imgenex).
F ab 2 fragment goat anti human igm
Manufactured by Jackson ImmunoResearch
F(ab')2 fragment Goat Anti-Human IgM is a laboratory reagent used for the detection and analysis of human immunoglobulin M (IgM) in various immunological assays. It is prepared by pepsin digestion of goat anti-human IgM antibodies, resulting in the F(ab')2 fragment, which retains the antigen-binding capability but lacks the Fc region.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
B cell isolation kit, by Miltenyi Biotec (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Perm buffer 3, by BD (1 mentions)
Fix buffer 1, by BD (1 mentions)
Anti erk1 2 pt202 py204, by BD (1 mentions)
Anti iκbα, by BD (1 mentions)
Megacd40l, by Enzo Life Sciences (1 mentions)
2 protocols using f ab 2 fragment goat anti human igm
1
Primary B Cell Activation and Lymphoma Treatment
2
Activated B Cell Signaling Assay
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Frozen PBMCs were thawed the day before the assay and cultivated overnight in complete medium. PBMCs were first stained for dead cells and washed, and 5 × 105 PBMCs were distributed in a 96-well V-bottom plate. Cells were stimulated or not stimulated with complete medium containing 100 ng/ml Mega CD40L (Enzo Life Sciences), 20 µg/ml F(ab′)2 fragment goat anti–human IgM (Jackson ImmunoResearch Laboratories, Inc.), or 40 ng/ml PMA. Optimal stimulation time points for each readout were selected based on prior kinetic experiments performed on control PBMCs. After 5 (p-ERK1/2 measurement)- or 35 (p-P65 and IκBα measurement)-min stimulation, cells were fixed by adding Fix Buffer I (1:1 volume; BD) and incubated 10 min at 37°C. Cells were then permeabilized for 20 min at room temperatures using Perm Buffer III (BD) and stained for 3 h at room temperature with anti-CD20 (H1; BD) and IgG2b isotypic control, anti–NF-κB P65-(pS259) (BD), anti-ERK1/2 pT202/pY204 (BD), or anti-IκBα (BD). Cells were subsequently acquired on a FACS Gallios flow cytometer and analyzed with FlowJo software (v10).
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