To induce B-cell receptor crosslinking, OSU-CLL and MEC-1 were treated with 10 µg/mL of goat F(ab’)2 anti-human IgM (Jackson ImmunoResearch, West Grove, PA, USA) for the final 15 min of treatment. Cells were then harvested and lysed for immunoblot analyses.
F ab 2 goat anti human igm
Manufactured by Jackson ImmunoResearch
Sourced in United States
F(ab)2 goat anti-human IgM is a laboratory reagent used for the detection and quantification of human IgM antibodies. It is produced by pepsin digestion of goat-derived anti-human IgM antibodies, resulting in the F(ab)2 fragment. This fragment retains the antigen-binding capability of the original antibody but lacks the Fc region.
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Lab products found in correlation
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Cpg odn, by InvivoGen (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Annexin 5, by BD (2 mentions)
Leaf puri ed anti cd3 and anti cd28 antibodies, by BioLegend (2 mentions)
Leaf puri ed fasl, by BioLegend (2 mentions)
7 aad antibodies, by BD (2 mentions)
Penicillin streptomycin, by Sartorius (1 mentions)
Nf kb p65, by Cell Signaling Technology (1 mentions)
Fetal calf serum (fcs), by Sartorius (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
L glutamine, by Sartorius (1 mentions)
Dulbecco s phosphate buffered saline, by Sartorius (1 mentions)
Roswell park memorial institute (rpmi), by Sartorius (1 mentions)
Zap70, by Cell Signaling Technology (1 mentions)
Cd79a, by Cell Signaling Technology (1 mentions)
Phospho shp1 tyr564, by Cell Signaling Technology (1 mentions)
Purified anti human actin antibody, by MP Biomedicals (1 mentions)
Goat anti rabbit igg h l hrp conjugate and goat anti mouse igg h l hrp conjugate, by Jackson ImmunoResearch (1 mentions)
Dimethyl sulfoxide dmso from, by Merck Group (1 mentions)
14 protocols using f ab 2 goat anti human igm
1
Inducing B-cell Receptor Crosslinking
2
Multiparameter Immune Cell Analysis
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ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), Akt (pan), phospho-Akt (S473), CD79b, CD79a, phospho-CD79a (Tyr182), SHP1, phospho-SHP1 (Tyr564), SHIP1, phospho-SHIP1 (Tyr1020), BTK, phospho-BTK(Tyr223), NF-kB p65, phospho–NF-kB p65 (ser536), IgM, Lyn, phospho-Lyn (Y507), LCK, CD86, and ZAP70 antibodies were from Cell Signaling Technology (Beverly, MA). Anti-SRC family (phospho-Y418)–phospho-Lyn (Y396), CD62L, and IgG antibodies were from Abcam (Cambridge, UK). Purified anti-human actin antibody was obtained from MP Biomedicals (Illkirch, France). Goat anti Rabbit IgG (H+L)–HRP conjugate and Goat anti Mouse IgG (H+L)–HRP conjugate, Goat F(ab′)2 anti-human IgM and Goat F(ab′)2 anti-human IgG were from Jackson Immunoresearch Laboratories (West Grove, PA). All antibodies utilized in the study were used in concentrations according to the manufacturer’s instructions. Ficoll-Paque PLUS from GE healthcare (Uppsala, Sweden), dimethyl sulfoxide (DMSO) from Merck (Darmstadt, Germany), RPMI, fetal calf serum (FCS), Dulbecco’s phosphate-buffered saline (PBS), L-glutamine, and penicillin-streptomycin from Biological Industries (Beit-Haemek, Israel) were utilized for cell cultures.
3
Regulation of IL-10 and Apoptosis in CD5+ B Cells
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Splenocytes were stimulated with 10 µg/mL of affinity purified polyclonal goat F(ab’)2 anti-human IgM (Jackson ImmunoResearch) for 24 h at 37°C in the presence or absence of humanized anti-human FasL-neutralizing mAb (RNOK203) or the general caspase inhibitor Z-VAD-FMK (BD Biosciences). During the last 6 h of incubation, cultures were stimulated with PMA and ionomycin in the presence of Golgi-Plug, followed by surface staining and intracellular detection of IL-10 and active caspase 3 using specific mAbs. It is noteworthy that number of cells obtained per sample was limiting and did not allow a more direct and extensive analysis such as multi-color FACS analysis for FasL, Fas, CD5 on B cells without culture with anti-FasL mAb. Such analysis would have directly addressed whether IL-10-producing CD5+ B cells are specifically targeted for apoptosis.
4
Stimulating Tumor Microenvironment Signals
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SpiD3, SpiD7, and analog 19 were synthesized at University of Nebraska Medical Center (UNMC; Omaha, NE) following reported procedures (13–15 (link)). Thapsigargin, cycloheximide, z-VAD(OMe)-FMK, ibrutinib, and JQ-1 were purchased from Cayman Chemical. TPCA-1 was purchased from Sigma-Aldrich. All pharmacologic agents/inhibitors were dissolved in DMSO. Stimulants used to mimic TME signals included 20 ng/mL TNFα (Cayman Chemical), 500 ng/mL recombinant human (rh) sCD40 ligand (Peprotech), 50 ng/mL rhBAFF ligand (Peprotech), 10 µg/mL goat F(ab)2 anti-human IgM (Jackson ImmunoResearch), 3.2 µmol/L CpG oligonucleotides 2006 (CpG; Integrated DNA Technologies), 1X lipopolysaccharide (Invitrogen), and 1X phorbol 12-myristate 13-acetate/Ionomycin (BioLegend).
5
Phosphorylation of B Cell Signaling Intermediates
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Five unrelated healthy adult volunteers were studied, each on a different day. Peripheral blood B cells were isolated as described in the Cell purification and cell culture conditions for in vitro treatment section. For phosphorylation assays, glucocorticoid- or vehicle-treated B cells were stained with mAbs against CD19, CD27, CD10, and IgD, as detailed in the Assessment of B cell surface markers section, then stimulated with 10 µg/ml goat F(ab′)2 anti-human IgM (Jackson ImmunoResearch Laboratories) at 37°C for 2 min. For the detection of phosphorylated signaling intermediates, cells were fixed and permeabilized using BD Cytofix and Phosflow Perm/Wash buffers (BD Biosciences) and stained separately with a PE-conjugated mAb against phosphorylated PLC-γ2 (pY759) clone K86-689.37 (BD Biosciences), or an unconjugated polyclonal antibody against phosphorylated CD79A(Tyr182) (Cell Signaling Technology; cat. no. 5173) followed by staining with secondary goat anti-rabbit IgG-PE (Thermo Fisher; cat. no. P-2771MP). Flow cytometric analyses were performed as described above.
6
BCR and CD40 Crosslinking Assay
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For BCR crosslinking 1 × 107 cells were treated with goat anti‐human IgM F(ab′)2 (Jackson ImmunoResearch) at 10 μg/mL final concentration for 8‐24 hours. CD40 ligand (R&D Systems) was used at 50 ng/mL final concentration for 1 hour.
7
Naïve B Cell Activation and Phenotyping
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Sorted naive B cells (1 × 105 in 200 μL per well) were plated and activated with goat anti-human IgM F(ab′)2 (10 μg/mL, Jackson ImmunoResearch), recombinant human CD40L (500 ng/mL, Enzo Life Sciences), IL-21 (100 ng/mL, Gibco, Thermo Fisher Scientific), IFN-γ (50 ng/mL, R&D Systems, Bio-Techne), IL-4 (25 ng/mL, R&D Systems, Bio-Techne), and IL-17 (50 ng/mL, R&D Systems, Bio-Techne). Cells were cultured in complete RPMI and incubated at 37°C for 5 days. Cells were harvested and stained with CD19, CD38, T-bet, CD27, CD11c, IL-21R, and CD71 antibodies before acquisition on a cytometer.
8
Cell stimulation and activation assay
9
BCR-Induced Calcium Mobilization Assay
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106 DG-75 or Ramos B cells were loaded in 700 µl RPMI containing 5% FCS, 1 µM Indo1-AM (Molecular Probes), and 0.015% Pluronic F127 (Molecular Probes) at 30°C for 25 min. Subsequently, the cell suspension was diluted two-fold with RPMI 10% FCS and incubated for 10 min at 37°C. Cells were washed, treated with 10 µM 1G244 (or mock-treated) and prepared for measurements as described earlier (Stork et al., 2007 (link)). For BCR-induced Ca2+ mobilization, cells were treated with 10 µg/ml F(ab)2 goat-anti-human IgM (Jackson Immunoresearch). Changes in the ratio of fluorescence intensities at 405 nm and 510 nm were monitored on a LSRII flow cytometer (Becton Dickinson) and analysed with FlowJo (TriStar).
10
Quantifying Protein Expression in Activated B Cells
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CpG or LPS pre-treated HBL1 B cells were placed on six-well culture plates previously coated overnight with 100 μg/mL of F(ab′)2 goat anti-human IgM (Jackson Immunoresearch) diluted in cold PBS and then processed to be analyzed by western blot. Band intensity was determined by ImageJ. Normalization of protein expression was performed by calculating the density of the target protein, multiplied by the ratio between the density of the loading control from the control sample (time 0 or untreated) and the density of loading control from the lane of interest. The fold change was calculated by dividing the normalized expression from each lane by the normalized expression of the control sample.
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