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Goat f ab 2 anti igm

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Goat F(ab′)2 anti-IgM is a reagent used for the detection and quantification of immunoglobulin M (IgM) in various immunoassays. It is prepared from the F(ab′)2 fragment of goat anti-IgM antibodies, which specifically bind to the Fc region of IgM molecules.

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Lab products found in correlation

Anti cd40, by Thermo Fisher Scientific (2 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) Muramyl dipeptide, by InvivoGen (1 mentions) C12 ie dap, by InvivoGen (1 mentions) Anti cd69 pe, by Thermo Fisher Scientific (1 mentions) Cytofix and phosflow perm wash buffers, by BD (1 mentions) Pe conjugated mabs, by BD (1 mentions) Recombinant murine il 4, by Thermo Fisher Scientific (1 mentions)

5 protocols using goat f ab 2 anti igm

1

CD69 Expression and B Cell Activation

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For CD69 analysis, cell sorter–purified B cells were cultured in a 96-well U-plate at 2 × 105 cells in 150 µl medium per well, with or without stimulation. Stimulating reagents were C12-iE-DAP (InvivoGen) at 10 µg/ml, goat F(ab′)2 anti-IgM (Jackson Laboratories) at 10 µg/ml, LPS (Sigma-Aldrich) at 20 µg/ml, CpG (InvivoGen) at 3 µg/ml, anti-CD40 (eBioscience) at 5 µg/ml, and muramyl dipeptide (InvivoGen) at 10 µg/ml. 6 h after stimulation, CD69 expression was analyzed using PE–anti-CD69 (eBioscience). Unseparated spleen cells were also stimulated using the same conditions, then costained with reagents to define various cell types together with anti-CD69. For survival analysis after culture, total cell number, viability, and cell size were measured by flow cytometry. BAFF (Alexis) was used at 0.2 µg/ml. For dye dilution cell division analyses, purified B cells were stained with CSFE (BioLegend), followed by stimulation in culture.
Hayakawa K., Formica A.M., Zhou Y., Ichikawa D., Asano M., Li Y.S., Shinton S.A., Brill-Dashoff J., Núñez G, & Hardy R.R. (2017). NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome. The Journal of Experimental Medicine, 214(10), 3067-3083.
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2

BCR Signaling Pathway Activation

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Freshly isolated PBMCs (1X106) in RPMI-1640 medium were allowed to rest at 37° C in CO2 incubator for 30 min before stimulation. BCR signaling was induced by crosslinking the BCR (Wang et al., 2014 (link)) with 10 μg/mL goat F(ab′) 2 anti-IgM (Jackson ImmunoResearch) at 37° C in CO2 incubator for indicated time points. Time course analysis was achieved by adding anti-IgM to each sample in reverse time points and fixing all samples in unison. To determine basal levels of phosphorylation, parallel cultures of unstimulated PBMCs were fixed at time zero. To detect phosphorylated CD79a (pIgα), cells were fixed (1.5% paraformaldehyde, 5 min, room temperature), permeabilized (90% methanol, 10 min, 4° C), and stained with rabbit antibodies specific for pCD79A (Igα, Tyr82) followed by PE–conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories).
Ahmed R., Omidian Z., Giwa A., Cornwell B., Majety N., Bell D.R., Lee S., Zhang H., Michels A., Desiderio S., Sadegh-Nasseri S., Rabb H., Gritsch S., Suva M.L., Cahan P., Zhou R., Jie C., Donner T, & Hamad A.R. (2019). A Public BCR Present in a Unique Dual-Receptor-Expressing Lymphocyte from Type 1 Diabetes Patients Encodes a Potent T Cell Autoantigen. Cell, 177(6), 1583-1599.e16.
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3

Activation of Murine B Cells

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Spleen single-cell suspensions were depleted of red blood cells and cultured in RPMI media supplemented with 10% FCS, 2μM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin. Cells were stimulated with goat F(ab′)2 anti-IgM (10 μg/mL, Jackson Immunoresearch) for 6h, and upregulation of activation markers was assessed by flow cytometry.
Cuenca M., Romero X., Sintes J., Terhorst C, & Engel P. (2015). Targeting of Ly9 (CD229) disrupts marginal zone and B1 B cell homeostasis and antibody responses. Journal of immunology (Baltimore, Md. : 1950), 196(2), 726-737.
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4

Flow Cytometry Analysis of BCR Signaling

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B cells stained with flow cytometry mAbs to CD20, CD27, CD21, IgG3, and IgD (identified above) were stimulated with 10 μg/ml goat F(ab’)2 anti-IgM (Jackson ImmunoResearch Laboratories; Cat# 109–006-129) 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 were stained separately with PE-conjugated mAbs to Syk phosphorylated at Tyr348 (clone I120–722), Btk phosphorylated at Tyr223 (clone N35–86) and PLC-γ2 phosphorylated at Tyr759 (clone K86–689.37) (all from BD Biosciences). Flow cytometry was performed on an LSRFortessa.
Kardava L., Sohn H., Youn C., Austin J.W., Wang W., Buckner C.M., Justement J.S., Melson V.A., Roth G.E., Hand M.A., Gittens K.R., Kwan R.W., Sneller M.C., Li Y., Chun T.W., Sun P.D., Pierce S.K, & Moir S. (2018). IgG3 regulates tissue-like memory B cells in HIV-infected individuals. Nature immunology, 19(9), 1001-1012.
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5

Stimulation of ATA B1a and Arrested B Cells

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Cell sorter purified ATA B1a cells (B220+CD5+IgMb–) in peritoneal cavity (pB1) and arrested cells (B220+AA4+IgMb–) in spleen in ATA μκTg mice were cultured in a 96-well U-plate at 2 × 105 cells in 150 µl medium (with 10% FCS)/well (in duplicate), with or without stimulation. Stimulating reagents: LPS (Sigma-Aldrich) at 20 µg/ml, CpG (InvivoGen) at 3 µg/ml, goat F(ab')2 anti-IgM (The Jackson Laboratory) at 10 µg/ml, anti-CD40 (eBioscience) at 5 µg/ml, and recombinant murine IL-4 (PeproTech) at 10 ng/ml.
Hayakawa K., Formica A.M., Brill-Dashoff J., Shinton S.A., Ichikawa D., Zhou Y., Morse HC I.I.I, & Hardy R.R. (2016). Early generated B1 B cells with restricted BCRs become chronic lymphocytic leukemia with continued c-Myc and low Bmf expression. The Journal of Experimental Medicine, 213(13), 3007-3024.
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