Cd20 magnetic beads

Manufactured by Miltenyi Biotec

Sourced in Cameroon

CD20 magnetic beads are a laboratory product from Miltenyi Biotec. They are designed for the magnetic separation and isolation of CD20-positive cells from various sample types.

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Magnetic beads (547 citations) Anti-CD20 magnetic beads (2 citations)

6 protocols using cd20 magnetic beads

Purification and Characterization of B Cells

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Peripheral B cells were purified from the blood of patients and HD by Ficoll density gradient purification followed by positive selection using CD20 magnetic beads (Miltenyi Biotec). Bone marrow and splenic B cells from humanized mice were enriched using anti-human CD19 magnetic beads (Miltenyi Biotec). Purified B cells were then stained with the following antibodies: anti-CD10 (clone: HI10A), anti-CD19 (clone: HIB19), anti-CD21 (clone: B-LY4), anti-CD27 (clone: O323), anti-CD34 (clone: 581), anti-CD45 (clone: HI30), anti-CD69 (clone: FN50), anti-CD86 (clone: IT2.2), anti-CXCR4 (clone: 12G5), anti-IgM (clone: MHM88), anti-IgD (clone: IA6–2), anti-PD-1 (clone: EH12.2H7), anti-RANKL (clone: MIH24) (all from Biolegend), anti-CD3 (clone: OKT3) and 7AAD (eBioscience), and annexin V (AF488 conjugated) (ThermoFisher Scientific). Intracellular staining was performed with anti-p-ATM (clone: 10H11.E12) and anti-γ-H2AX (clone: 2F3) (Biolegend) after staining for surface markers using a fixation-permeabilization solution kit (eBioscience). Flow cytometry was performed using a BD LSRII, and the data were analyzed using Flow Jo software (Treestar).

Mensah K.A., Chen J.W., Schickel J.N., Isnardi I., Yamakawa N., Vega-Loza A., Anolik J.H., Gatti R.A., Gelfand E.W., Montgomery R.R., Horowitz M.C., Craft J.E, & Meffre E. (2019). Impaired ATM Activation in B Cells is Associated with Bone Resorption in Rheumatoid Arthritis. Science translational medicine, 11(519), eaaw4626.

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Isolation and Characterization of Human B Cell Subsets

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B cells were purified from PBMCs by positive selection using CD20 magnetic beads (Miltenyi Biotec, Cambridge, MA). Single CD19⁺CD10⁺IgM^hiCD21^loCD27⁻ new emigrant/transitional and CD19⁺CD10⁻IgM⁺CD21⁺CD27⁻ mature naive B cells were sorted on a FACSAria flow cytometer (BD Biosciences, San Jose, CA) into 96 well PCR plates. Reverse transcription of cDNA, RT-PCR reactions, primer sequences, cloning strategy, expression vectors, antibody expression, antibody purification, and recombinant antibody reactivity determination were performed as previously described ^{7 (link)}. A highly polyreactive antibody (ED38) ^{7 (link), 20 (link)} was used as a positive control in HEp-2 reactivity and polyreactivity ELISAs. Antibodies were considered polyreactive when they recognized all 3 analyzed antigens, which included dsDNA, insulin, and lipopolysaccharide (LPS). The reactivity of purified recombinant antibodies was also tested on HEp-2 cell coated slides (Bion Enterprise, LTD, Des Plaines, IL) by indirect immunofluorescence.

Janssen E., Morbach H., Ullas S., Bannock J.M., Massad C., Menard L., Barlan I., Lefranc G., Su H., Dasouki M., Al-Herz W., Keles S., Chatila T., Geha R.S, & Meffre E. (2014). DOCK8 deficient patients have a breakdown in peripheral B cell tolerance and defective regulatory T cells. The Journal of allergy and clinical immunology, 134(6), 1365-1374.

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Comprehensive B and T Cell Phenotyping

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B cells were purified from the patient’s peripheral blood by positive selection using CD20 magnetic beads (Miltenyi Biotec, Cambridge, MA). CD4⁺ T cells were isolated from the peripheral blood of research subjects using the EasySep human CD4+ T cell enrichment kit (STEMCELL Technologies, Cambridge, MA). The following antibodies were used for flow cytometric staining: CD19 APC-Cy7, CD27 PerCP-Cy5.5, CD10 PE-Cy7, CD21 V450, CD69 PE, CD86 APC, FAS Alexa 647, CD4 APC-Cy7, CD127 PerCP-Cy5.5, CD45RO Alexa 700, CXCR5 PerCP-Cy5.5, PD-1 PE-Cy7, ICOS APC (all from Biolegend, San Diego, CA), CD3 eFluor 605NC (from eBioscience, San Diego, CA), and CD21 BD Horizon V450 (BD). Intracellular staining for FOXP3 Alexa 488 (eBioscience) and T-bet PE was performed using the FOXP3/Transcription Factor Staining Buffer Set (eBioscience) in accordance with the manufacturer’s directions.

Minto H., Mensah K.A., Reynolds P.R., Meffre E., Rubtsova K, & Gelfand E.W. (2019). A Novel ATM Mutation Associated with Elevated Atypical Lymphocyte Populations, Hyper-IgM, and Cutaneous Granulomas. Clinical immunology (Orlando, Fla.), 200, 55-63.

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Sorting and Phenotyping of B Cell Subsets

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Peripheral B cells were purified from the blood of patients and control donors by positive selection using CD20 magnetic beads (Miltenyi Biotec). For sorting, enriched B cells were stained with FITC-anti-IgM, PE-anti-IgG, PE-Cy7-anti-CD10, APC-anti-CD21, Pacific Blue-anti-CD19, and PercP-Cy5.5-anti-CD27 (all from BioLegend) (gating strategy is shown in fig. S21). Single- or batch-sorted CD19⁺CD21^loCD10⁺IgM^hiCD27⁻ new emigrant and CD19⁺CD21⁺CD10⁻IgM⁺CD27⁻ mature naïve B cells from patients, heterozygous relatives, and HDs were sorted on a FACSAria flow cytometer (Becton Dickinson, Mountain View, CA) into 96-well polymerase chain reaction (PCR) plates or 5-ml round-bottom polystyrene test tubes, respectively. For phenotyping, enriched B cells were stained with FITC-anti-IgM, PE-anti-CD69, PE-Cy7-anti-CD10, APC-anti-CD86, PercP-Cy5.5-anti-CD27, APC-Cy7-anti-CD19 (all from BioLegend), and V450-anti-CD21 (BD Bioscience) (gating strategy is shown in fig. S22).

Sng J., Ayoglu B., Chen J.W., Schickel J.N., Ferre E.M., Glauzy S., Romberg N., Hoenig M., Cunningham-Rundles C., Utz P.J., Lionakis M.S, & Meffre E. (2019). AIRE expression controls the peripheral selection of autoreactive B cells. Science immunology, 4(34), eaav6778.

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Isolation and Sorting of Transitional and Naive B Cells

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Single B cells were isolated as previously described.16 Briefly, peripheral B cells from the MG patients and healthy controls were isolated by positive selection using CD20 magnetic beads (Miltenyi). Single CD19⁺CD21^loCD10⁺IgM^hiCD27^– new emigrant/transitional and CD19⁺CD21⁺CD10^–IgM⁺CD27^– mature naive B cells were sorted on a FACSAria flow cytometer (BD) into 96‐well PCR plates and immediately frozen on dry ice.

Lee J., Stathopoulos P., Gupta S., Bannock J.M., Barohn R.J., Cotzomi E., Dimachkie M.M., Jacobson L., Lee C.S., Morbach H., Querol L., Shan J., Vander Heiden J.A., Waters P., Vincent A., Nowak R.J, & O'Connor K.C. (2016). Compromised fidelity of B‐cell tolerance checkpoints in AChR and MuSK myasthenia gravis. Annals of Clinical and Translational Neurology, 3(6), 443-454.

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Purifying and Stimulating B Cells

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B cells were purified from the peripheral blood by positive selection using CD20 magnetic beads (Miltenyi Biotec) and plated for 48 hours at 150,000-200,000 cells per well in a 96-well plate in RPMI containing 10% FBS alone or RPMI+10% FBS and either 2 μg/ml polyclonal F(ab')2 rabbit anti-human IgM (Jackson ImmunoResearch, West Grove, PA), multimeric, soluble, 0.05 μg/ml recombinant-human CD40L (Alexis Biochemicals, San Diego, CA), 2 μg/ml gardiquimod (TLR7 agonist; Invitrogen, Waltham, MA), or 0.5 μg/ml CpG (TLR9 agonist; Invitrogen).

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