Anti b220 antibody

Manufactured by BioLegend

Sourced in United States

The Anti-B220 antibody is a laboratory reagent used for the identification and detection of the B220 surface antigen, also known as CD45R. It is commonly used in flow cytometry, immunohistochemistry, and other immunoassays to label and identify B cells in various biological samples.

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Lab products found in correlation

Magnisort mouse b cell enrichment kit, by Thermo Fisher Scientific (2 mentions) Anti cd40 hm40 3 agonistic antibody, by Thermo Fisher Scientific (2 mentions) Mil 4, by R&D Systems (2 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) Facscalibur, by BD (2 mentions) Fluoview version 1.7c software, by Olympus (2 mentions) Anti cd20, by Abcam (2 mentions) Vectashield mounting medium, by Vector Laboratories (2 mentions) Fv1000 confocal microscope, by Olympus (2 mentions) Cellquest, by BD (1 mentions) Anti cd19, by Cell Signaling Technology (1 mentions) Anti cd20, by Cell Signaling Technology (1 mentions) Ab65407, by Thermo Fisher Scientific (1 mentions) Tgf β, by Thermo Fisher Scientific (1 mentions) Retinoic acid, by Merck Group (1 mentions) Cellquest software, by BD (1 mentions)

Close spelling variants of this product

Alexa Fluor 647 anti-mouse B220 antibody (clone RA3–6B2, PE-conjugated anti-mouse B220 antibody Anti-B220

4 protocols using anti b220 antibody

Purification and Stimulation of Murine Splenic B Cells

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Mouse splenic B cells were purified by negative selection (eBioscience Magnisort Mouse B cell enrichment kit). B-cell purity was >95% as measured by FACS analysis (FACSCalibur and CellQuest software; BD Biosciences) using anti-B220 antibody (BioLegend). Purified B cells were seeded at a final concentration of 0.5 or 0.25 × 10⁶ cells/mL. For plasmablast differentiation, B cells were stimulated with 5 µg/mL LPS (Sigma) for 72 h, and for IgG1 CSR, B cells were stimulated 5 µg/mL anti-CD40 (HM40-3) agonistic antibody (eBioscience), or 5 µg/mL LPS (Sigma), together with 5 ng/mL mIL-4 (R&D Systems) for 96 h. All B cells were cultured in RPMI 1640 supplemented with 10% (vol/vol) heat-inactivated FCS, 5 mM Hepes, 2 mM l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM 2-mercaptoethanol.

Chiu H., Mallya S., Nguyen P., Mai A., Jackson L.V., Winkler D.G., DiNitto J.P., Brophy E.E., McGovern K., Kutok J.L, & Fruman D.A. (2017). The Selective Phosphoinoside-3-Kinase p110δ Inhibitor IPI-3063 Potently Suppresses B Cell Survival, Proliferation, and Differentiation. Frontiers in Immunology, 8, 747.

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Double Immunofluorescence Staining of B-Cells

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Double immunofluorescence staining was performed as previously described.^{36 (link)} Briefly, B-cells were fixed in 4% paraformaldehyde (15 min, RT), washed with PBS, permeabilized (0.1% Triton X-100 in PBS, 15 min), and attached on pre-coated poly-L-lysine slides. Slides were blocked in 3% BSA, followed by mouse monoclonal anti-NS3 (Abcam; 1:100, ab65407) or anti-core (ThermoFisher Scientific, Waltham, MA, USA; 1:100, MA1-080) antibody staining. The cells were further incubated with rabbit polyclonal anti-CD19, anti-CD20 (Cell Signaling Technology, Inc., Danvers, MA, USA; 1:400; 3574), anti-CD20 (Abcam; 1:400; ab78237) or anti-B220 antibody (Biolegend, San Diego, CA, USA; 1:200; #103201). Cells were washed with PBS and incubated with goat anti-rabbit Alexa Fluor 488 and goat anti-mouse or anti-rat Alexa Fluor 597 (Invitrogen; 1:200). The stained slides were mounted using Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA, USA). Control slides were similarly processed, except primary antisera were omitted, which yielded no staining. Images were captured using an Olympus FV1000 confocal microscope and processed with Olympus Fluoview Version 1.7c software (Olympus Corporation, Tokyo, Japan).

Dai B., Chen A.Y., Corkum C.P., Peroutka R.J., Landon A., Houng S., Muniandy P.A., Zhang Y., Lehrmann E., Mazan-Mamczarz K., Steinhardt J., Shlyak M., Chen Q.C., Becker K.G., Livak F., Michalak T.I., Talwani R, & Gartenhaus R.B. (2015). Hepatitis C virus upregulates B-cell receptor signaling: a novel mechanism for HCV-associated B-cell lymphoproliferative disorders. Oncogene, 35(23), 2979-2990.

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Immunostaining of Hepatitis C Virus Proteins

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Double immunofluorescence staining was performed as previously described.^{36 (link)} Briefly, B cells were fixed in 4% paraformaldehyde (15 min, room temperature), washed with phosphate-buffered saline, permeabilized (0.1% Triton X-100 in phosphate-buffered saline, 15 min) and attached on precoated poly-L-lysine slides. Slides were blocked in 3% bovine serum albumin, followed by mouse monoclonal anti-NS3 (Abcam; 1:100, ab65407) or anti-core (ThermoFisher Scientific; 1:100, MA1-080) antibody staining. The cells were further incubated with rabbit polyclonal anti-CD19, anti-CD20 (Cell Signaling Technology, Inc., Danvers, MA, USA; 1:400; 3574), anti-CD20 (Abcam; 1:400; ab78237) or anti-B220 antibody (Biolegend; 1:200; #103201). Cells were washed with phosphate-buffered saline and incubated with goat anti-rabbit Alexa Fluor 488 and goat anti-mouse or anti-rat Alexa Fluor 597 (Invitrogen; 1:200). The stained slides were mounted using Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA, USA). Control slides were similarly processed, except primary antisera were omitted, which yielded no staining. Images were captured using an Olympus FV1000 confocal microscope and processed with Olympus Fluoview Version 1.7c software (Olympus Corporation, Tokyo, Japan).

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Induction of Immunoglobulin Class Switching in Murine B Cells

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Splenic B cells were purified by negative selection (eBioscience Magnisort Mouse B cell enrichment kit). B cell purity measured by FACS analysis (FACSCalibur and CellQuest software; BD Biosciences) using anti-B220 antibody (Biolegend) yielded >95% B220+. Purified B cells were seeded at a final concentration of 1.5 × 10^6 cells per milliliter. For class switching experiments, B cells were stimulated 5 μg/mL anti-CD40 (HM40–3) agonistic antibody (eBioscience), or 5 μg/mL LPS (Sigma), together with 5 ng/mL mIL-4 (R&D Systems) for 96 h to induce switching to IgG1. 5 μg/mL LPS (Sigma) induced switching to IgG3. 5 μg/mL LPS (Sigma) with 5 ng/mL mIL-4 (R&D), 5 ng/mL mIL-5 (Tonbo Bioscience), 5 nM retinoic acid (Sigma Aldrich) and 5 ng/mL TGF-β (Thermo Fisher Scientific) induced switching to IgA. All cells were cultured with RPMI 1640 supplemented with 10% (vol/vol) heat-inactivated FCS, 5 mM Hepes, 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, 50 μM 2-mercaptoethanol.

Chiu H., Jackson L.V., Oh K.I., Mai A., Ronai Z.A., Ruggero D, & Fruman D.A. (2018). The mTORC1/4E-BP/eIF4E Axis Promotes Antibody Class Switching in B Lymphocytes. Journal of immunology (Baltimore, Md. : 1950), 202(2), 579-590.

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