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Anti mouse igm f ab 2

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Anti-mouse IgM F(ab')2 is a reagent used in immunological research. It is a fragment of an antibody that specifically binds to mouse IgM antibodies.

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Lab products found in correlation

Cypher5e, by GE Healthcare (2 mentions) Biotinylated anti cd23, by BD (2 mentions) Il 21, by Thermo Fisher Scientific (2 mentions) L glutamine, by Corning (2 mentions) Rpmi 1640 medium, by Corning (2 mentions) Non essential amino acids (neaa), by Corning (2 mentions) Streptavidin microbeads, by Miltenyi Biotec (2 mentions) Glutamax, by Thermo Fisher Scientific (2 mentions) Anti b220 apc, by Thermo Fisher Scientific (1 mentions) Cpg 1668, by InvivoGen (1 mentions) Pe donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions) Anti mouse igg, by Merck Group (1 mentions) Opd substrate, by Merck Group (1 mentions) Live dead fixable far red dye, by Thermo Fisher Scientific (1 mentions) Peroxidase conjugated anti mouse igg, by Merck Group (1 mentions) Celltrace cfse, by Thermo Fisher Scientific (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Phosphatase inhibitor cocktail, by Nacalai Tesque (1 mentions) Protease inhibitor cocktail, by Nacalai Tesque (1 mentions) Anti iκbα antibody, by Cell Signaling Technology (1 mentions)

11 protocols using anti mouse igm f ab 2

1

Stimulation of Lymph Node B Cells

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Single cell suspensions were generated from lymph nodes of 8–10 week old mice, resuspended at 2×107 cells/ml, rested in serum free media at 37°C for 1 hour, and stimulated in 96 well V-bottom plates at 37°C with vehicle, CpG 1668 (Invivogen) or F(ab’)2 anti-mouse IgM (Jackson Immunoresearch) for the times indicated in the figure legend. Cells were fixed with pre-warmed 1% paraformaldehyde, washed, permeabilized with ice cold 100% methanol, washed, rehydrated in PBS, stained with antibodies against pERK or IκBα (Cell Signaling Technologies), washed, stained with PE-donkey anti-rabbit IgG (Jackson Immunoresearch) and anti-B220-APC (eBiosciences), and analyzed as described above.
Mills R.E., Lam V.C., Tan A., Cresalia N., Oksenberg N., Zikherman J., Anderson M., Weiss A, & Hermiston M.L. (2015). Unbiased modifier screen reveals that signal strength determines the regulatory role murine TLR9 plays in autoantibody production. Journal of immunology (Baltimore, Md. : 1950), 194(8), 3675-3686.
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2

Efferocytosis Assay for B Cells

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CD23+ B cells were purified from single cell suspensions of splenocytes with biotinylated anti-CD23 (BD Bioscience; #553137) and streptavidin microbeads (Miltenyi Biotec; #130-048-101) according to manufacturer’s instructions. Cells were cultured for 2–3 d in RPMI 1640 medium (Corning) supplemented with non-essential amino acids (Corning), 2 mM l-Glutamine (Corning), 25 mM HEPES (pH 7.2–7.6), and 50 μM β-Mercaptoethanol and stimulated with 5 μg/mL F(ab’)2 anti-mouse IgM (Jackson ImmunoResearch), 5 μg/mL purified anti-mouse CD40 (BioXcell), and 50 ng/mL IL-21 (Peprotech). For thymocyte engulfment assays68 (link) thymocytes were harvested and isolated from 4–6 week-old WT mice and treated with 50 μM Dexamethasone for 4 h to induce apoptosis. Apoptotic thymocytes were then stained with 1 μM CypHer5E (GE Healthcare) for 45 min at 37 oC in serum-free Hank’s Balanced Sodium Solution (HBSS). Stained thymocytes were co-cultured with splenocytes at a 1:10 splenocyte to thymocyte ratio. Apoptotic thymocytes were removed by washing with cold PBS and splenocytes were assessed for efferocytosis by flow cytometry. MHC-II expression was compared on efferocytic (CypHer5E+) and non-efferocytic (CypHer5E−) B cells.
Ricker E., Manni M., Flores-Castro D., Jenkins D., Gupta S., Rivera-Correa J., Meng W., Rosenfeld A.M., Pannellini T., Bachu M., Chinenov Y., Sculco P.K., Jessberger R., Prak E.T, & Pernis A.B. (2021). Altered function and differentiation of age-associated B cells contribute to the female bias in lupus mice. Nature Communications, 12, 4813.
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3

BALB/c Mice Antibody Response Assay

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BALB/c mice received i.p. 250 μg of Ly9.7.144 or IgG1 control mAb. Twenty-four hours later, spleen lymphocytes were isolated and stained with CellTrace™ CFSE (Life Technologies) following manufacturer’s instructions. A total of 106 lymphocytes were cultured in 0.5 mL of complete RPMI in 24-well plates. Cells were cultured for 72h in medium alone or in the presence of 10 μg/mL LPS (Sigma), or 10 μg/mL F(ab′)2 anti-mouse IgM (Jackson ImmunoResearch). Viability was assessed by staining with Live/Dead Fixable far red dye (Invitrogen) following the manufacturer’s protocol.
IgG levels in the supernatants were determined by ELISA. 96-well plates were coated with 3 μg/mL anti-mouse IgG (Sigma). A purified in-house mouse IgG antibody was used as a standard. Bound IgG was detected with peroxidase-conjugated anti-mouse IgG (Sigma). The reaction was developed with OPD substrate (Sigma) and read at 405 nm.
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

Immunoblot Analysis of Splenic B Cell Signaling

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Purified 5 × 105 splenic B cells were suspended in 50 μl serum free RPMI 1640 medium. After pre-incubation for 10 min at 37°C, the cells were stimulated with F(ab′)2 anti-mouse IgM (10 μg per 5 × 105 cells) (Jackson ImmunoResearch, West Grove, PA, USA) at 37°C. The stimulated B cells were lysed in NP-40 lysis buffer containing 1% NP-40, 50 mM Tris–HCl, 5 mM EDTA, 150 mM NaCl, phosphatase inhibitor cocktail and protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Cell lysates were resolved by SDS–PAGE and transferred to PVDF membranes. Then, the blots were probed with the indicated antibodies, followed by HRP-conjugated secondary antibodies and were visualized using Luminata Forte Western HRP Substrate (Merck Millipore, Darmstadt, Germany). The following antibodies were used for immunoblotting: anti-phospho-tyrosine antibodies 4G10 (Merck Millipore, Burlington, MA, USA); anti-PLCγ2 antibodies (sc407, Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-IκBα antibodies (4814); and anti-Erk1/2 antibodies (4695, Cell Signaling Technology, Danvers, MA, USA). Immunoblots were visualized using the Bio-Rad ChemiDoc MP imaging system (Bio-Rad).
Sasanuma H., Ozawa M, & Yoshida N. (2018). RNA-binding protein Ptbp1 is essential for BCR-mediated antibody production. International Immunology, 31(3), 157-166.
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5

Efferocytosis and MHC-II Expression in B Cells

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CD23 + B cells were purified from single cell suspensions of splenocytes with biotinylated anti-CD23 (BD Bioscience) and streptavidin microbeads (Miltenyi) according to manufacturer's instructions. Cells were cultured for 2-3d in RPMI 1640 medium (Corning) supplemented with non-essential amino acids (Corning), 2mM L-Glutamine (Corning), 25mM HEPES (pH 7.2-7.6), and 50µM b-Mercaptoethanol and stimulated with 5µg/mL F(ab')2 anti-mouse IgM (Jackson ImmunoResearch), 5µg/mL purified anti-mouse CD40 (BioXcell), and 50ng/mL IL21 (Peprotech). Thymocyte engulfment assays were performed as previously described 68 . In brief, thymocytes were harvested and isolated from 4-6 week-old WT mice and treated with 50µM Dexamethasone for 4hr to induce apoptosis. Apoptotic thymocytes were then stained with 1µM CypHer5E (GE Healthcare) for 45min at 37 o C in serum-free Hank's Balanced Sodium Solution (HBSS). Stained thymocytes were co-cultured with splenocytes at a 1:10 splenocyte to thymocyte ratio. Apoptotic thymocytes were removed by washing with cold PBS and splenocytes were assessed for efferocytosis by flow cytometry. MHC-II expression was compared on efferocytic (CypHer5E + ) and non-efferocytic (CypHer5E -) B cells.
Ricker E., Manni M., Flores-Castro D., Jenkins D.E., Gupta S., Rivera‐Correa J., Meng W., Rosenfeld A.M., Pannellini T., Bachu M., Chinenov Y., Sculco P.K., Jessberger R., Prak E.T.L., & Pernis A.B. (2021). Sex-specific differences in the function and differentiation of ABCs mark TLR7-driven immunopathogenesis.
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6

Murine C5a: A Comprehensive Immunological Assay

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Murine C5a was from Cell Sciences (Canton, ME). Anti-mouse C3a and C5a mAbs were from BD Biosciences (San Jose, CA). C3ar1-A and C5ar1-A were purchased from Calbiochem (EMB Biochemicals). CFSE was used according to the manufacturer’s instructions (Invitrogen). Anti-mouse IgM F(ab’)2 was purchased from Jackson Labs (Bar Harbor, ME). FITC anti-mouse CD19, anti-mouse CD40, PE anti-mouse CD40, anti-mouse IL-6, biotin-anti-mouse IL-6, anti-mouse C5a, biotin anti-mouse C5a, BAFF, APRIL, PE anti-mouse TACI, and APC anti-mouse BAFF-R were purchased from BD Biosciences (San Jose, CA). HRP anti-mouse IgG was purchased from Cell Signal Technology (Danvers, MA) Recombinant mouse IL-4 was from Miltenyi Biotech, (San Diego, CA). and LPS (Escherichia coli O26:B6) from Sigma-Aldrich (St. Louis, MO)
Paiano J., Harland M., Strainic M.G., Nedrud J., Hussain W, & Medof M.E. (2019). Follicular B2 cell activation and Class Switch Recombination depend on autocrine C3ar1/C5ar1 signaling in B2 cells. Journal of immunology (Baltimore, Md. : 1950), 203(2), 379-388.
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7

B Cell Isolation and Stimulation

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B cells from the spleen were enriched by MACS, collecting the negative fraction of CD43 (Ly48) Microbeads (Miltenyi Biotec). Cells were cultured in RPMI 1640 (Corning) supplemented with 10% FBS (MilliporeSigma), 1x Penicillin-Streptomycin, 1x MEM Nonessential Amino Acids (Corning), 1mM Sodium Pyruvate, 2mM GlutaMax, and 55μM 2-Mercaptoethanol (Thermo Fisher Scientific). The following concentrations were used for cell stimulation: 10μg/ml anti-mouse IgM F(ab’)2 (Jackson ImmunoResearch), 10ng/ml mouse rBaff (R&D Systems), 5μg/ml anti-mouse CD40 (1C10) and 10ng/ml mouse rIL4 (Thermo Fisher Scientific). Total BM cells were cultured in IMDM with GlutaMax (Thermo Fisher Scientific) supplemented with 10% FBS (MilliporeSigma), 1x Penicillin-Streptomycin (Corning), 55μM 2-Mercaptoethanol (Thermo Fisher Scientific) and in the presence of 10ng/ml mouse rIL7 (PrepoTech).
Ramezani-Rad P., Leung C.R., Apgar J.R, & Rickert R.C. (2020). E3 ubiquitin ligase Fbw7 regulates the survival of mature B cells. Journal of immunology (Baltimore, Md. : 1950), 204(6), 1535-1542.
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8

B Cell Proliferation Assay Using Mouse Splenocytes

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We isolated enriched B cell fractions from mouse spleens using the MagniSort® Mouse B cell Enrichment Kit (Life Technologies, #8804–6827-74), according to manufacturer instructions. Purified B cells (>95% purity) were washed with cold PBS and labeled with 10 uM eF450 Cell proliferation dye (eBioscience, #65–0842-85) for 10 minutes at 37°C. The reaction was then blocked by adding cold B cell medium (RPMI, supplemented with 10% FBS, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 50 uM β-mercaptoethanol; Life Technologies) and incubating 5 minutes on ice. Cells were washed and resuspended in B cell medium at 1×10^6/mL, plated in 12 or 6-well plates and stimulated using different cytokine/ligand combinations, as detailed in the legends. Specifically: anti-CD40 (12.5 ng/mL, clone HM-40–3, Life Technologies); mouse IL-4 (20 ng/mL, Peprotech); anti-mouse IgM F(ab)2 (3 ug/mL; Jackson ImmunoResearch); Lipopolysaccharides from Escherichia coli O55:B5 (1 ug/mL, Sigma-Aldrich); or anti-mouse CD180 (RP105) (1 ug/mL, Biolegend). In all cases, we stimulated the cells for 72 hours before analysis. Dilution of eF450-CPD was assessed by flow cytometry using a BD Fortessa cytometer (BD Bioscience) and data analyzed with FlowJo software, Mac version v10.5.3 (Flowjo, LLC - BD).
Roberto M.P., Varano G., Vinas-Castells R., Holmes A.B., Kumar R., Pasqualucci L., Farinha P., Scott D.W, & Dominguez-Sola D. (2021). Mutations in the transcription factor FOXO1 mimic positive selection signals to promote germinal center B cell expansion and lymphomagenesis. Immunity, 54(8), 1807-1824.e14.
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9

Activation and Viability of Splenic B Cells

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Splenic B cells were purified by negative selection of CD43+ cells using anti-CD43 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). The purified B cells (1 × 107 cells/ml) were stimulated with 10 µg/mL anti-mouse IgM F(ab)′2 (Jackson ImmunoResearch, West Grove, PA, USA) or 10 µg/mL anti-human IgM F(ab)′2 (Jackson ImmunoResearch) and then lysed in lysis buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100 and 0.5 mM EDTA plus protease and phosphatase inhibitor cocktails (Nacalai Tesque)]. Samples were transferred to polyvinylidene difluoride membranes by electrophoresis and analysed by immunoblotting with antibodies against p-Syk (Tyr525/526) (C87C1) and Syk (D3Z1E) (Cell Signaling Technology, Danvers, MA, USA). The purified splenic B cells were cultured with or without 25 ng/ml BAFF (R&D Systems, Minneapolis, MN, USA) for 24, 48, or 72 h. The frequency of live B cells was assessed using TOPRO3 (Invitrogen) exclusion.
Satofuka H., Abe S., Moriwaki T., Okada A., Kazuki K., Tanaka H., Yamazaki K., Hichiwa G., Morimoto K., Takayama H., Nakayama Y., Hatano S., Yada Y., Murakami Y., Baba Y., Oshimura M., Tomizuka K, & Kazuki Y. (2022). Efficient human-like antibody repertoire and hybridoma production in trans-chromosomic mice carrying megabase-sized human immunoglobulin loci. Nature Communications, 13, 1841.
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10

Single-cell Splenic Immune Cell Stimulation

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Single-cell suspensions were obtained from untreated mouse spleens and rested in the supplemented RPMI 1640 at 37°C for an hour before stimulation. Cells were then stimulated with nicotine for 20 min and subsequently anti-mouse IgM F(ab′)2 (Jackson ImmunoResearch) for 2 min (pCD19) or 5 min (pAkt). Stimulation was ceased by adding prewarmed Lyse/Fix buffer (BD Biosciences). Fixed cells were then incubated with Perm/Wash buffer (BD Biosciences) for 30 to 60 min and stained with antibodies listed in table S1.
Kurata-Sato I., Mughrabi I.T., Rana M., Gerber M., Al-Abed Y., Sherry B., Zanos S, & Diamond B. (2024). Vagus nerve stimulation modulates distinct acetylcholine receptors on B cells and limits the germinal center response. Science Advances, 10(17), eadn3760.
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