Anti b220 magnetic beads

Manufactured by Miltenyi Biotec

Anti-B220 magnetic beads are a laboratory tool used for the isolation and enrichment of B220-positive cells. These beads are coated with antibodies that specifically bind to the B220 surface antigen, allowing for the separation and purification of B cells from complex cell mixtures using magnetic separation techniques.

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Close spelling variants of this product

Magnetic beads (547 citations) Anti-B220 beads (6 citations) Anti-B220 conjugated magnetic beads (3 citations) B220 magnetic beads (2 citations) Anti-mouse B220+ magnetic beads (2 citations)

6 protocols using anti b220 magnetic beads

Isolation and Stimulation of Murine B and T Cells

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B cells were isolated from murine spleens using anti-B220 magnetic beads per manufacturer’s protocol (Miltenyi Biotec) with >98% purity achieved. B cells were washed and then cultured in triplicate at 5 × 10⁴ cells/mL in RPMI 1640 + 10% fetal bovine serum, with 25 μg/mL LPS (E. coli 0111:B4, Calbiochem) with or without recombinant mouse IL-4 (10 ng/mL, Life Technologies)⁸⁰. Cell counts were performed at day 3 of stimulation using BioRad TC20 automated cell counter. Supernatants were collected for measurement of immunoglobulins (see ELISA section above), or LDH release per manufacturer’s protocol (Pierce LDH Cytotoxicity Assay Kit, ThermoScientific). A subset was stained with CFSE [5-(and 6)-carboxyfluorescein diacetate succinimidyl ester] per manufacturer’s protocol (eBioscience, Inc.).
T cells were isolated from murine spleens using Pan T-cell isolation magnetic beads per manufacturer’s protocol (Miltenyi Biotec) with >98% purity achieved. T cells were washed and then cultured at 10⁶ cells/mL in RPMI 1640 + 10% fetal bovine serum and stimulated with 20 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 μg/mL ionomycin. Supernatants were collected for measurement of IL-2 (see ELISA section above).

Broderick L., Yost S., Li D., McGeough M.D., Booshehri L.M., Guaderrama M., Brydges S.D., Kucharova K., Patel N.C., Harr M., Hakonarson H., Zackai E., Cowell I.G., Austin C.A., Hügle B., Gebauer C., Zhang J., Xu X., Wang J., Croker B.A., Frazer K.A., Putnam C.D, & Hoffman H.M. (2019). Mutations in topoisomerase IIβ result in a B cell immunodeficiency. Nature Communications, 10, 3644.

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Flow Cytometry Analysis of Immune Cells

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Monoclonal antibodies were purchased from Thermo Fisher Scientific eBioscience or BD Bioscience. Clones used were: anti-CD4 (RM4-5, dilution 1:500), anti-CD8α (53–6.7 dilution 1:500), anti-TCRβ (H57-597, dilution 1:250), anti-HSA (M1/69, dilution 1:500), anti-CD69 (H1.2F3, dilution 1:250), anti-CD44 (IM7, dilution 1:500), anti-CD62L (MEL-14, dilution 1:500), anti-CD19 (ID3, dilution 1:300), anti-CD127 (SB/199, dilution 1:250), anti-Caspase-3 (C92-605, 10 μl of Ab per test). Cell proliferation dye E450 was obtained from Thermo Fisher Scientific eBioscience. After staining with antibodies and DAPI (Thermo Fisher Scientific), cells were analyzed with an LSRII flow cytometer (BD Biosciences) or sorted with an Aria II (BD Biosciences). Post sort sample purity was >98%. In most cases, anti-CD4, anti-CD8, anti-PE, and anti-B220 magnetic beads (Miltenyi) were used for enrichment and depletion on MACS columns (Miltenyi) before sorting. Flow cytometry data were analyzed using Flowjo software (Tree Star).

Issuree P.D., Day K., Au C., Raviram R., Zappile P., Skok J.A., Xue H.H., Myers R.M, & Littman D.R. (2018). Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development. Nature Communications, 9, 3594.

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Isolation and Activation of Resting B Cells

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For B cells isolation, resting B cells were purified from spleen by negative selection with anti-CD43 magnetic beads or positive selection with anti-B220 magnetic beads (MiltenyiBiotech) following the manufacturer’s instructions. The purified B cells population was >95% B220 positive cells. B cells were cultured in RPMI supplemented with 10% FCS, 2-ME, penicillin (100 U/ml), and streptomycin (100 μg/ml). Purified B cells (2×10⁶/ml) were cultured with LPS (10 μg/ml) or IgM-specific goat F(ab’)2 antibody (10 μg/ml) in a 48-well flat-bottom plate.

Meng X., Grötsch B., Luo Y., Knaup K.X., Wiesener M.S., Chen X.X., Jantsch J., Fillatreau S., Schett G, & Bozec A. (2018). Hypoxia-inducible factor-1α is a critical transcription factor for IL-10-producing B cells in autoimmune disease. Nature Communications, 9, 251.

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Cre-mediated Recombination Validation

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Cre-mediated recombination of R26^lsl.Btg1Q36H and R26^lsl.Btg1wt alleles was validated on gDNA extracted from sorted naïve B cells and germinal center B cells or from B220⁺-enriched B cells as indicated, using Puregene Gentra cell kit (QIAGEN 158388). B220⁺ B cells were enriched by positive selection with anti-B220 magnetic beads (Miltenyi Biotec 130-049-501). We performed PCR on gDNA using primers that specifically amplified the recombined targeted allele and Phusion HF polymerase (NEB M0530). The PCR program was 98°C for 30 s, [98°C for 15 s, 70°C for 30 s, and 72°C for 30 s] for 30 cycles, and 72°C for 10 min. PCR products were resolved by agarose gel electrophoresis and visualized on a ChemiDoc Touch imaging system (BioRad) using SYBR Safe DNA stain (Thermo Fisher S33102). The same primers were used to confirm expression of Cre-recombined alleles on total RNA extracted from B220⁺-enriched B cells, reverse transcribed, and analyzed by qRT-PCR. Forward primer annealed to the Rosa26 short homology arm upstream of the cassette and reverse primer to the kozak sequence and Btg1 coding sequence from the R26^lsl.Btg1Q36H or R26^lsl.Btg1wt at the Rosa26 locus (fig. S2, A and D), to ensure only recombined targeted alleles and transcripts were amplified from gDNA and total RNA, respectively. Primer details are included in table S8.

Sutcliffe J.G., Foye P.E., Erlander M.G., Hilbush B.S., Bodzin L.J., Durham J.T, & Hasel K.W. (2000). TOGA: An automated parsing technology for analyzing expression of nearly all genes. Proceedings of the National Academy of Sciences of the United States of America, 97(5), 1976-1981.

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In vitro B cell differentiation

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To mimic TI immunization in vitro, splenic B cells were purified from β1^WT and β1^KO CD21cre Prdm1^+/gfp mice using anti-B220 magnetic beads (Miltenyi Biotec) and cultured with 25 μg/ml LPS (L5668; Sigma-Aldrich). After 4 d, three populations were differentiated: CD138⁻Blimp⁻ Act B cells, CD138⁻Blimp⁺ (pre-PB), and CD138⁺Blimp⁺ (PB). To differentiate CD138⁺Blimp⁺ cells under TD conditions, B220⁺ cells were cultured for 5 d in the presence of CD40L (5 ng/ml), IL-4, and IL-5 (10 ng/ml; Peprotech).

Andreani V., Ramamoorthy S., Fässler R, & Grosschedl R. (2022). Integrin β1 regulates marginal zone B cell differentiation and PI3K signaling. The Journal of Experimental Medicine, 220(1), e20220342.

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Splenic B Cell Differentiation Assay

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To mimic TI immunization in vitro, splenic B cells were purified from Mzb1^+/+ and Mzb1^−/−Prdm1^+/gfp mice using anti-B220 magnetic beads (Miltenyi Biotec) and cultured with 25 μg/mL LPS (L5668; Sigma-Aldrich). After 4 d, three populations were isolated: CD138⁻ Blimp⁻ activated B (Act B) cells, CD138⁻ Blimp⁺ (Pre-PB), and CD138⁺Blimp⁺ (PB). In some experiments cells were differentiated in 96-U–bottom-well plates, coated overnight at 4 °C with VCAM-1 (2.5, 5 and 10 µg/mL; R&D Systems). After coating, plates were washed and blocked with Iscove’s modified Dulbecco’s medium (IMDM)-BSA 1% for 1 h at 37 °C. B220⁺ cells (1 × 10⁵) were added to the wells and cultured with LPS, in the absence or presence of recombinant CXCL12 (0.5 µg/mL; R&D Systems). To differentiate CD138⁺Blimp⁺ cells under TD conditions, B220⁺ cells were cultured for 5 d in the presence of CD40L (5 ng/mL), IL-4, and IL-5 (10 ng/mL; Peprotech).

Andreani V., Ramamoorthy S., Pandey A., Lupar E., Nutt S.L., Lämmermann T, & Grosschedl R. (2018). Cochaperone Mzb1 is a key effector of Blimp1 in plasma cell differentiation and β1-integrin function. Proceedings of the National Academy of Sciences of the United States of America, 115(41), E9630-E9639.

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