B220 ra3 6b2

Manufactured by Thermo Fisher Scientific

Sourced in United States

The B220 (RA3-6B2) is a lab equipment product designed for cell surface marker detection. It functions as an antibody reagent for the specific identification and enumeration of B220-positive cells.

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45 protocols using b220 ra3 6b2

Multiparametric Phenotyping of Immune Cells

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Single-cell suspensions of spleen, bone marrow, and peritoneal cavity were obtained as previously described (1 (link)) and stained using fluorochrome or biotin-conjugated antibodies against B220 (RA3-6B2), IgM (μ-chain, Life Technology), IgMb (AF6-78), IgD (11-26c.2a), CD5 (53–7.3), CD11b (M1/70), CD11c (HL3), F4/80 (BM8, eBioscience), Ly6G (IA8), CD19 (ID3), CD21 (7G6), CD23 (B3B4), CD93 (AA4.1), CD86 (GL1), CD44 (IM7), CD43 (S7), CD9 (KMC8) and/or CD138 (281-2). Unless otherwise stated, antibodies were procured from BD Biosciences. Biotin-conjugated antibodies were secondarily stained with streptavidin-conjugated fluorochromes and dead cells were excluded using fixable viability dye 455UV or eFluor 450 (eBioscience) or Alexa Fluor 700-conjugated succinimidyl ester (Life Technologies). For intracellular staining, cells were fixed using 1.6% paraformaldehyde (Electron Microscopy Sciences), then permeabilized with a solution of 0.05% Triton-X-100 (SigmaUltra) and stained with rabbit anti-mouse BTK (D3H5, Cell Signaling), followed by a fluorochrome-conjugated anti-Rabbit IgG (F′ab2) secondary (Cell Signaling). Samples were collected on an LSRII flow cytometer (BD Biosciences) and data analyzed using FlowJo software (TreeStar).

Nyhoff L.E., Clark E.S., Barron B.L., Bonami R.H., Khan W.N, & Kendall P.L. (2018). Bruton’s tyrosine kinase is not essential for B cell survival beyond early developmental stages. Journal of immunology (Baltimore, Md. : 1950), 200(7), 2352-2361.

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Comprehensive Panel of Anti-Murine Antibodies

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Anti-murine antibodies include: B220 (RA3-6B2), CD138 (281-2), CD21 (7G6), CD24 (M1/69), CD80 (16-10A1), CD86 (GL1), CD5 (53-7.3) from BD Biosciences; CD19 (ID3), CD44 (IM7), CD21 (7E9), CD23 (B3B4), CD24 (M1/69), TACI (1A1) from BioLegend; CD11c (N418), TACI (8F10-3), AA4.1 (AA4.1), AID (mAID-2), T-bet (4B10), CD11b (M1/70) from eBioscience; B220 (RA3-6B2) from Life Technologies; TACI (166010) from R&D Systems; goat anti-mouse IgM-, IgG-, IgA-, IgG1-, IgG2b-, IgG2c-, IgG3-HRP conjugated, unlabeled, or isotype, IgG2c (1079-02) from Southern Biotechnology.

Jacobs H.M., Thouvenel C.D., Leach S., Arkatkar T., Metzler G., Scharping N.E., Kolhatkar N.S., Rawlings D.J, & Jackson S.W. (2016). BAFF promotes autoantibody production via TACI-dependent activation of transitional B cells. Journal of immunology (Baltimore, Md. : 1950), 196(9), 3525-3531.

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Modulating Androgen Levels in Mice

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Degarelix (as acetate), a third generation LHRH-Ant (Firmagon), was resuspended in sterile water for injection and administered s.c. to mice at a dose of 40 µg/g. Lupron (11.25-mg 3-mo depot), an LHRH-Ag, was prepared according to the manufacturer’s instructions and administrated intramuscularly to mice at a dose of 20 µg/g. Degarelix and Lupron were purchased from the MSKCC Pharmacy. Testosterone propionate (Sigma-Aldrich) was resuspended in peanut oil and injected daily s.c. (1 mg/mouse) in 100 µl. Surface antibodies against CD44 (IM7), EpCAM (G8.8), PDGFRa (APA5), PECAM-1 (390), CD45 (30-F11), and H-2Kk (AF3-12.1.3) were purchased from eBioscience; anti-Ly-51 (BP-1), CD34 (RAM34), CD62L (MEL-14), H-2Kb (AF6-88.5), IFN-γ (XMG1.2), IL-2 (JES6-5H4), c-Kit (2B8), CD3ε (145-2C11), CD25 (PC61), TER-119 (TER-110), and CD8α (53–6.7) were purchased from BD; anti-CD4 (RM4-5) and B220 (RA3-6B2) were purchased form Invitrogen; anti-CD44 (IM7), CD90.2 (30-H12), and IA/IE (M5/114.15.2) were purchased from BioLegend; and Ulex europaeus agglutinin 1 (UEA-1) was purchased from Vector Laboratories. For in vitro cultures of T cell differentiation (Fig. 2, A and B), cells were stained with antibodies to lineage (Lin)-specific markers: CD8, CD11c, NK1.1, CD3, CD4, B220, CD11b, and GR-1.

Velardi E., Tsai J.J., Holland A.M., Wertheimer T., Yu V.W., Zakrzewski J.L., Tuckett A.Z., Singer N.V., West M.L., Smith O.M., Young L.F., Kreines F.M., Levy E.R., Boyd R.L., Scadden D.T., Dudakov J.A, & van den Brink M.R. (2014). Sex steroid blockade enhances thymopoiesis by modulating Notch signaling. The Journal of Experimental Medicine, 211(12), 2341-2349.

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Immunofluorescent Staining of Frozen Spleen

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Spleens were snap frozen in Tissue Tek (Sakura Finetek). Frozen sections (8 μm) were fixed with acetone permeabilized with 0.05% saponin. The following antibodies were used for the staining: CD11c (N418) from BioLegend (Ref 117301), p-eIF2α (Ser 52) from Invitrogen (Ref 44-728G), CD11b (M1/70) from BD Biosciences, CD8α-biotin (53-6.7) BioLegend (Ref 100703), and B220 (RA3-6B2) from Invitrogen (Ref 14-0452-81). Images were collected using a Zeiss LSM 510 confocal microscope. Image processing was performed with Zeiss LSM software.

Mendes A., Gigan J.P., Rodriguez Rodrigues C., Choteau S.A., Sanseau D., Barros D., Almeida C., Camosseto V., Chasson L., Paton A.W., Paton J.C., Argüello R.J., Lennon-Duménil A.M., Gatti E, & Pierre P. (2020). Proteostasis in dendritic cells is controlled by the PERK signaling axis independently of ATF4. Life Science Alliance, 4(2), e202000865.

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Multiparameter Flow Cytometry of Tumor Cells

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Tumor tissues were digested both mechanically by chopping with razor blades and chemically with 1mg/mL type IA collagenase (Sigma-Aldrich) for 30 minutes at 37°C. Following digestion, cell suspensions were washed, filtered and stained as previously described (O’Sullivan et al., 2012 (link)). The following antibodies were used: Ly6C (ER-MP20, Serotec), MHCII (M5/114 15.2, eBioscience), Ly6G (1A8, Biolegend), CD8 (53-6.7, eBioscience), CD44 (IM7, Biolegend), CD3 (17A.2, Biolegend), CD4 (GK1.5, Biolegend), CD69 (H1.2F3, Biolegend), Granzyme B (NGZB, eBioscience), IFNγ (XMG 1.2, Biolegend), TCRβ (H57-597, Biolegend), B220 (RA3-6B2, eBioscience), NK1.1 (PK136, Biolegend), CD11b (M1/70, eBioscience), CD45 (30-F11, Biolegend). Stained cell suspensions were analyzed on a BD FACS CANTO II (BD Biosciences).

Saddawi-Konefka R., Seelige R., Gross E.T., Levy E., Searles S.C., Washington A J.r., Santosa E.K., Liu B., O’Sullivan T.E., Harismendy O, & Bui J.D. (2016). Nrf2 induces IL-17D to mediate tumor and virus surveillance. Cell reports, 16(9), 2348-2358.

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Multiparameter Flow Cytometry Analysis

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Fluorochrome-conjugated antibodies against mouse CD45 (30-F11), CD3 (145-2C11), CD8 (53-6.7), B220 (RA3-6B2), CD11b (M1/70), CD11c (N418), Gr1 (RB6-8C5), TER119 (TER119), NKp46 (29A1.4), CD4 (RM4-5), Thy1.2 (53-2.1), SCA1 (D7), CCR6 (29-2L17), c-Kit (2B8), IL-7Rα (A7R34), IL-22 (1H8PWSR), IL-17A (TC11-18H10.1), IFNγ (XMG1.2), T-bet (4B10), RORγt (2B2), EpCAM (G8.8) were purchased from eBioscience, BD Pharmingen, or Biolegend. For intracellular cytokine staining, cells were stimulated with IL-23 (50 ng/ml) in the presence of brefeldin A (10 µg/ml) for 4 hours at 37°C. Cell sorting and flow cytometry were performed using an Influx cell sorter and a LSRII flow cytometer (BD Biosciences), respectively. Data were analyzed using FlowJo vX software (TreeStar).

Macho-Fernandez E., Koroleva E.P., Spencer C.M., Tighe M., Torrado E., Cooper A.M., Fu Y.X, & Tumanov A.V. (2014). Lymphotoxin beta receptor signaling limits mucosal damage through driving IL-23 production by epithelial cells. Mucosal immunology, 8(2), 403-413.

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Quantifying Peritoneal Macrophage Subsets

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Two methods were used to quantify peritoneal macrophages. Total peritoneal cells were counted in a hemacytometer or using an automated cell counter. Then this number was multiplied by the percent of macrophages stained for CD115 (AFS98; eBioscience), ICAM-2 (3C4; BioLegend) and F4/80 (BM8, eBioscience) by flow cytometry. These antibodies allow us to distinguish the minor and major peritoneal macrophage populations (Gautier et al., 2012 (link)), with only the major macrophage population expressing ICAM-2 (Gautier et al., 2012 (link)). Alternatively, peritoneal cells obtained by lavage were incubated with FITC-conjugated anti–ICAM-2 mAb, and then a manual count of the fraction of ICAM-2⁺ cells was made and multiplied by the total number of peritoneal cells. Both methods yielded similar results. Other reagents and mAbs used in flow cytometry were Annexin V (Miltenyi Biotec) and antibodies against Ki67 (B56; BD), active caspase 3 (C92-605; BD), pH3 (D2C8; Cell Signaling technology), TIMD4 (RMT4-54; eBioscience), CD206 (MR5D3; Serotec), LYVE-1 (Abcam), Siglec F (E50-2440; BD), CD11b (M1/70; eBioscience), CD5 (53–7.3; BD), B220 (RA3-6B2; eBioscience), ly 6C/G (RB6-8C5; eBioscience), Ly6-G (1A8; BD), TCRb (H57-597; eBioscience), MHC-II (M5/114.15.2; eBioscience), and MARCO (ED31; Serotec).

Gautier E.L., Ivanov S., Williams J.W., Huang S.C., Marcelin G., Fairfax K., Wang P.L., Francis J.S., Leone P., Wilson D.B., Artyomov M.N., Pearce E.J, & Randolph G.J. (2014). Gata6 regulates aspartoacylase expression in resident peritoneal macrophages and controls their survival. The Journal of Experimental Medicine, 211(8), 1525-1531.

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Multiparametric Flow Cytometry Analysis

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Ears were collected and processed as described^{54 (link)}. All cells were first pre-incubated with anti-mouse CD16/CD32 for blockade of Fc γ receptors, then were washed and incubated for 40 min with the appropriate monoclonal antibody conjugates in a total volume of 200 μl PBS containing 2 mM EDTA and 2% (vol/vol) bovine serum. DAPI (Invitrogen) was used to distinguish live cells from dead cells during cell analysis. Stained cells were analyzed on a FACS Canto or LSRII machine using the Diva software (BD Bioscience). Data were analyzed with FlowJo software (TreeStar). The following fluorochrome-conjugated anti-mouse antibodies were used at indicated dilutions: CD103 (2E7, 1:200), CD11c (N418, 1:200), CD24 (30-F11, 1:200), CD11b (M1/70, 1:200), MHC-II (M5/114.15.2, 1:400), CD45 (30-F11, 1:200), CD64 (X54-5/7.1, 1:200), Ly6C (HK1.4, 1:200), TCRb (H57-597, 1:200), CD3 (145-2C11, 1:200), TCRgd (EBIOGL3, 1:200), B220 (RA3-6B2, 1:200), CD49b (DX5, 1:200) and FCeR1 (MAR-1, 1:200) were from eBioscience; Siglec-F (E50-2440, 1:200), Ly6G (1A8, 1:200), CD117 (2B8, 1:200), CD8 (53-6.7, 1:200), and CD4 (GK1.5, 1:200) were from BD Bioscience.

Chen L., Deshpande M., Grisotto M., Smaldini P., Garcia R., He Z., Gulko P.S., Lira S.A, & Furtado G.C. (2020). Skin expression of IL-23 drives the development of psoriasis and psoriatic arthritis in mice. Scientific Reports, 10, 8259.

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Immunophenotyping of Spleen and LN Cells

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Spleens and popliteal LNs were harvested at indicated times and prepared as previously described^{57 (link)}. Briefly, tissues were fixed in periodate-lysine-paraformaldehyde buffer and placed in 30% sucrose in PBS. Tissues were then embedded in OCT medium (Electron Microscopy Sciences), frozen in dry-ice cooled isopentane and sections were cut on a cryostat (Leica Microsystems). Sections were blocked in 5% sera and stained with the following antibodies to: CD11b (M1/70, eBioscience), CD11c (N418, eBioscience), B220 (RA3–6B2, eBioscience), CD64 (polyclonal, R&D Systems), MHC II (I-A/I-E, M5/114.15.2, eBioscience), followed by the relevant secondary antibodies conjugated to fluorophores. Images were acquired using Leica SP8 microscope and analyzed using Imaris software (Bitplane).

Baharom F., Ramirez-Valdez R.A., Tobin K.K., Yamane H., Dutertre C.A., Khalilnezhad A., Reynoso G.V., Coble V.L., Lynn G.M., Mulè M.P., Martins A.J., Finnigan J.P., Zhang X.M., Hamerman J.A., Bhardwaj N., Tsang J.S., Hickman H.D., Ginhoux F., Ishizuka A.S, & Seder R.A. (2020). Intravenous Nanoparticle Vaccination Generates Stem-Like TCF1+ Neoantigen-Specific CD8+ T Cells. Nature immunology, 22(1), 41-52.

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Immunophenotyping of Spleen and LN Cells

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