F ab 2 fragment of goat anti mouse igm

Manufactured by Jackson ImmunoResearch

Sourced in United States

The F(ab')2 fragment of goat anti-mouse IgM is a laboratory reagent used in various immunological and biochemical applications. It is prepared by pepsin digestion of goat-derived antibodies that specifically recognize mouse immunoglobulin M (IgM) molecules. The F(ab')2 fragment retains the antigen-binding capabilities of the original antibody while lacking the Fc region.

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3 protocols using f ab 2 fragment of goat anti mouse igm

Activation and Proliferation Assays for T and B Cells

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CD3⁺ (T cells) or B220⁺ (B cells) cells isolated (Miltenyi Biotec, Inc, per manufacturers’ directions) from WT or S5A splenocytes were incubated overnight with anti-CD3 (1 μg/ml; 145–2C11, eBioscience)/anti-CD28 (1 μg/ml; 37.51, eBioscience) (T cells) or plate-bound anti-IgM 10 μg/ml; F(ab’)2 fragment of goat anti-mouse IgM, Jackson ImmunoResearch, West Grove, PA) (B cells). Upregulation of CD69 and CD86 was assessed by flow cytometry (6 (link), 24 (link)).
For proliferation assays, CD3⁺ (T cells) or B220⁺ cells (B cells) isolated from WT or S5A splenocytes using negative selection (Miltenyi Biotec Inc.) and were labeled with CTV (Invitrogen). Cells were incubated for 72 h in the absence or presence of anti-CD3/anti-CD28 (T cells) or of soluble anti-IgM stimulation (10 μg/ml; F(ab’)2 fragment of goat anti-mouse IgM, Jackson ImmunoResearch, West Grove, PA), and rIL-4 (10 ng/ml, R&D Systems) (B cells). Cells were analyzed for CTV dilution by flow cytometry.

Anaya E.P., Lin X., Todd E.M., Szasz T.P, & Morley S.C. (2021). Novel mouse model reveals that serine phosphorylation of L-plastin is essential for effective splenic clearance of pneumococcus. Journal of immunology (Baltimore, Md. : 1950), 206(9), 2135-2145.

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Splenic B Cell Proliferation Assay

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Splenic B cells were purified by depleting CD43⁺ cells, using anti-CD43 beads and magnetic columns (Miltenyi Biotec, Bergisch Gladbach, Germany) and stimulated in vitro with 10 μg/ml F(ab’)2 fragment of goat anti-mouse IgM (Jackson ImmunoResearch, West Grove, USA) in combination with 25U/ml of recombinant mouse IL-4 (R&D Systems, Minneapolis, USA), 5μg/ml bacterial LPS (Sigma-Aldrich), or 5 μg/ml of bacterial LPS in combination with 25 U/ml of recombinant mouse IL-4. Labeling of cells with 5-(6-) carboxyfluorescein diacetate, succinimidyl ester (CFSE, Molecular Probes, Eugene, USA) for analysis of proliferation was performed following the manufacturer’s instructions. The decline in CFSE fluorescence as a measure of B cell proliferation was determined by FACS analysis.

Dobenecker M.W., Marcello J., Becker A., Rudensky E., Bhanu N.V., Carrol T., Garcia B.A., Prinjha R., Yurchenko V, & Tarakhovsky A. (2020). The catalytic domain of the histone methyltransferase NSD2/MMSET is required for the generation of B1 cells in mice. FEBS letters, 594(20), 3324-3337.

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Plasma Membrane Repair Regulation

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Freshly isolated B cells were incubated with SLO in either Ca²⁺-containing (+Ca²⁺) or Ca²⁺-free DMEM (−Ca²⁺) for 5 min at 4°C to allow binding. Cells were then warmed to 37°C for 5 min, immediately transferred to 4°C, and stained with the membrane impermeant dye PI. Cells were analyzed using a BD FACS Canto II flow cytometer and FlowJo software (Tree Star). The percentage of cells repaired in the presence of Ca²⁺ (+Ca²⁺) was determined by [%PI positive (−Ca²⁺) − %PI positive (+Ca²⁺)] × 100/%PI positive (−Ca²⁺).
To determine the effect of SM on PM repair, SM from Bacillus cereus (50 µM; Sigma-Aldrich) was included in the medium during the 37°C incubation.
To analyze the effect of BCR cross-linking on the efficacy of PM repair, surface BCR were bound with Fab fragments of goat anti–mouse IgM (10 µg/ml) that does not cross-link BCRs (−XL), F(ab')₂ fragment of goat anti–mouse IgM (10 µg/ml; Jackson ImmunoResearch Laboratories) that cross-links BCRs (+XL) for 20 min at 4°C, or biotinylated Fab goat anti–mouse IgM (10 µg/ml; Jackson ImmunoResearch Laboratories) for 20 min at 4°C plus streptavidin (5 µg/ml; Jackson ImmunoResearch Laboratories) at 4°C for 10 min for extensive cross-linking of BCRs. After washing off unbound antibodies/streptavidin, the cells were then treated with SLO (200 ng/ml) and analyzed by flow cytometry as described above.

Miller H., Castro-Gomes T., Corrotte M., Tam C., Maugel T.K., Andrews N.W, & Song W. (2015). Lipid raft–dependent plasma membrane repair interferes with the activation of B lymphocytes. The Journal of Cell Biology, 211(6), 1193-1205.

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