High affinity (NP4) and total (NP29) NP-specific serum antibody titers were measured as previously described.69 (link) In brief, Immulon 4 HBX Microiter Plates (Thermo Fisher Scientific) were coated with 10μg/mL of NP4-BSA or NP29-BSA and blocked with 5% BSA in PBS. Serum was then added followed by serial dilution. For the detection of influenza virus-specific antibodies, ELISA plates were coated with 175ng of influenza virus and blocked with 5% BSA in PBS. Serum was added to the plate beginning with a 1:25 dilution, followed by serial dilution. Abs were then detected with one of the following biotinylated Abs: goat anti-mouse IgM (Jackson Immunoresearch), goat anti-mouse IgG (Jackson Immunoresearch), goat anti-mouse IgG1 (Invitrogen), or goat anti-mouse IgE (Southern Biotech), followed by streptavidin-alkaline phosphatase (Vector Laboratories). Plates were developed using p-nitrophenyl phosphate (disodium salt) (Thermo Fisher Scientific) and read at l405nm on a Synergy H1 (BioTek Instruments).
Goat anti mouse igm
Manufactured by Jackson ImmunoResearch
Sourced in United States
Goat anti-mouse IgM is a secondary antibody used for the detection of mouse IgM antibodies in various immunological assays. It is produced by immunizing goats with purified mouse IgM and affinity-purifying the resulting antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg, by Jackson ImmunoResearch (6 mentions)
Alkaline phosphatase conjugated goat anti rabbit igg, by Jackson ImmunoResearch (3 mentions)
Neurobasal, by Thermo Fisher Scientific (2 mentions)
Igepal, by Merck Group (2 mentions)
Monoclonal mouse igg antibodies, by Enzo Life Sciences (2 mentions)
Phenol red, by Thermo Fisher Scientific (2 mentions)
Indo 1 am, by Thermo Fisher Scientific (2 mentions)
Powerload concentrate, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
Streptavidin alkaline phosphatase, by Vector Laboratories (1 mentions)
Goat anti mouse ige, by Southern Biotech (1 mentions)
P nitrophenyl phosphate, by Thermo Fisher Scientific (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Cellection dynabeads, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Anti cd11b, by BD (1 mentions)
Goat anti mouse iga, by Southern Biotech (1 mentions)
Goat anti mouse ig, by Southern Biotech (1 mentions)
Rmm 1, by BioLegend (1 mentions)
31 protocols using goat anti mouse igm
1
Measurement of NP-Specific Antibody Titers
2
Isolation and Culture of Murine Retinal Ganglion Cells
3
B Cell Stimulation and Protein Analysis
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B cells were resuspended to 2 × 107 cells/mL in modified HEPES-suffered saline (mHBS; 25 mM HEPES, pH 7.2, 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 1 mg/mL glucose, 2 mM glutamine, 1 mM pyruvate, 50 μM 2-mercaptoethanol). The cells (3 × 106 in 150 μL) were then stimulated with 20 μg/mL goat anti-mouse IgG (Jackson ImmunoResearch, #115-005-008) or goat anti-mouse IgM (Jackson ImmunoResearch, ##115-005-020) for the indicated times at 37°C. Reactions were stopped by adding cold PBS with 1 mM Na3VO4. The cells were then pelleted for 5 min at 640 RCF at 4°C and lysed in RIPA buffer (30 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Igepal (Sigma-Aldrich), 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA) with protease and phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL pepstatin A, 10 μg/mL soybean trypsin inhibitor, 25 mM β-glycerophosphate, 1 mM Na3MoO4, 1 mM Na3VO4). Protein concentrations were determined using the bicinchoninic acid assay (Thermo Fisher, #23225). Cell extracts were analyzed by immunoblotting.
4
Serum Antibody Measurement Protocol
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96-well microplate was coated with goat anti-mouse IgM (Jackson ImmunoResearch) or goat anti-mouse Ig (Southern Biotech) and blocked with 2% BSA. Mouse sera was added in 2-fold serial dilutions and incubated for 1 h at room temperature. Detection was achieved with biotinylated rat anti-mouse IgM (Biolegend, RMM-1), goat anti-mouse IgA, goat anti-mouse IgG1, goat anti-mouse IgG2b, goat anti-mouse IgG2c, goat anti-mouse IgG3, goat anti-mouse Kappa/Lambda (all Southern Biotech) or H-2D(d) monomer (folded with RGPGRAFVTI peptide, NIH Tetramer Facility). Biotinylated antibodies were then detected with streptavidin-horseradish peroxidase reagent (BD Biosciences), followed by development with TMB substrate. The developing reaction was stopped with 1M phosphoric acid and read at 450 nM.
Human protective antibodies were measured using the following kits according to the manufacturer’s instructions: anti-measles IgG (Serion Immunodiagnostica GmbH), anti-mumps IgG (Calbiotech), anti-rubella IgG (Phoenix Pharmaceuticals Inc), anti-tetanus toxoid IgG (Serion Immunodiagnostica GmbH). Total IgM, IgA, and IgG were measured by nephelometry by the clinical laboratory at the Hospital of the University of Pennsylvania.
Human protective antibodies were measured using the following kits according to the manufacturer’s instructions: anti-measles IgG (Serion Immunodiagnostica GmbH), anti-mumps IgG (Calbiotech), anti-rubella IgG (Phoenix Pharmaceuticals Inc), anti-tetanus toxoid IgG (Serion Immunodiagnostica GmbH). Total IgM, IgA, and IgG were measured by nephelometry by the clinical laboratory at the Hospital of the University of Pennsylvania.
5
B Cell Migration Assay Protocol
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Fo B cells purified by magnetic-activated cell sorting were activated with 5 µg/ml goat anti–mouse IgM (Jackson ImmunoResearch) for 24 h or rested overnight (unstimulated control). Various dilutions of recombinant mouse CCL21 (provided by the late I. Clark-Lewis) or CXCL13 (PeproTech) in 150 µl chemotaxis buffer (RPMI-1640 with 0.5% BSA and 20 mM Hepes) were added to the lower chambers of Transwell chemotaxis plates (96-well, 5-µm pore size; Corning). Cells were extensively washed in chemotaxis buffer and loaded into the upper chambers at 105 cells/well in 50 µl chemotaxis buffer and incubated for 3 h at 37°C. To enumerate B cell migration, cells were harvested from the bottom chambers, and B220+ cells were assessed by flow cytometry using a defined number of CaliBRITE beads (BD) as an internal reference. The migration index was calculated as described (Kara et al., 2013 (link)).
6
Anti-IgM Stimulation of Splenocytes
7
Splenocyte Stimulation Assay
8
Investigating c-Myc and GC B Cells
9
BAFF-Mediated B Cell Proliferation
10
BCR Signaling Pathway Activation
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Raji D1.3 B cells (3 × 106 in 100 μL mHBS) were stimulated at 37°C with 20 μg/mL goat anti-mouse IgM (Jackson ImmunoResearch, #115-005-020) to engage the D1.3 BCR. Ramos B cells were stimulated with 20 μg/mL donkey anti-human IgM (Jackson ImmunoResearch, #709-005-073). Reactions were stopped, and cells lysed, by adding 30 μL RIPA buffer (30 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Igepal (Sigma-Aldrich), 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA) with 3X protease and phosphatase inhibitors (3 mM phenylmethylsulfonyl fluoride, 30 μg/mL leupeptin, 3 μg/mL aprotinin, 3 μg/mL pepstatin A, 75 mM β-glycerophosphate, 3 mM Na3MoO4, 3 mM Na3VO4). After 15 min on ice with intermittent mixing, insoluble material was removed by centrifugation. Cell lysates (20 μg protein) were separated by SDS-PAGE and then analyzed by immunoblotting with antibodies that recognize the phosphorylated CD79 ITAMs (pCD79; Cell Signaling Technologies, #5173, 1:000; overnight at 4°C), human GMFγ (Proteintech, #13625-1-AP, 1:500; overnight at 4°C) or β-actin (Santa Cruz, #sc-47778, 1:5,000; 1 h at room temperature), followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad, #170-6515; 1:3,000) or goat anti-mouse IgG (Bio-Rad, #170-6516; 1:3,000). After ECL detection, blots were quantified and imaged using a Li-Cor C-DiGit imaging system.
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