B cell isolation kit 2

Manufactured by Miltenyi Biotec

Sourced in Germany, United States

The B Cell Isolation Kit II is a lab equipment product from Miltenyi Biotec designed for the isolation of B cells from various cell samples. The kit utilizes magnetic separation technology to efficiently isolate B cells from the starting material.

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Lab products found in correlation

Automacs pro separator, by Miltenyi Biotec (4 mentions) Ficoll hypaque, by GE Healthcare (4 mentions) Monocyte isolation kit 2, by Miltenyi Biotec (4 mentions) Cd4 t cell isolation kit, by Miltenyi Biotec (3 mentions) Automacs, by Miltenyi Biotec (3 mentions) Ficoll histopaque, by GE Healthcare (3 mentions) Chromium device, by 10x Genomics (2 mentions) Single cell 5 d j kit, by 10x Genomics (2 mentions) Nextseq 500 platform, by Illumina (2 mentions) Pe labeled cxcr5 microbeads, by Miltenyi Biotec (2 mentions) Cd27 microbeads, by Miltenyi Biotec (2 mentions) Facsaria 2, by BD (2 mentions) Facsverse device, by BD (2 mentions) Cd4 t cell isolation kit 2, by Miltenyi Biotec (2 mentions) Anti igd ab, by Southern Biotech (2 mentions) Cd19 allophycocyanin hib19, by BD (2 mentions) Cd38 pecy hb7, by BD (2 mentions) Cd27 pe m t271, by BD (2 mentions) Igd fitc ia6 2, by BD (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)

Close spelling variants of this product

MACS B cell isolation kit II Mouse Pan B Cell Isolation Kit II Human naive B-cell isolation kit II Miltenyi Pan B Cell Isolation Kit II MACS Human B Cell Isolation Kit II Pan B Cell Isolation Kit II Human naïve B cell isolation kit II Naive B Cell Isolation Kit II MojoSort Mouse Pan B Cell Isolation Kit II Human B cell isolation kit II

71 protocols using b cell isolation kit 2

Isolation of Tonsillar and Peripheral B-cells

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Primary human tonsillar mononuclear cells (TMCs) were isolated from palatine tonsils obtained from pediatric patients who underwent tonsillectomy due to tonsillar hyperplasia. Tonsillar B-cells (TBCs) were prepared as described previously [23 (link)]. Briefly, tonsils were cut into small pieces with a scalpel in phosphate-buffered saline (PBS) and passed through a 70 μm-pore-size cell strainer (Falcon, Wohlen, Switzerland). Then, TMCs were purified by density gradient centrifugation with Ficoll-Paque Premium (VWR international-GE Healthcare, Dietikon, Switzerland). TBCs were isolated from TMCs using the B-cell isolation kit II according to the instructions of the manufacturer (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity of isolation was always over 95% as assessed by flow cytometry.
Peripheral B-cells (PBCs) were isolated from blood form healthy donors and prepared as following. Buffycoats were diluted 5× with phosphate-buffered saline (PBS). Then, peripheral blood mononuclear cells (PBMCs) were purified by density gradient centrifugation with Ficoll-Paque Premium (VWR international-GE Healthcare). PBCs were isolated from PBMCs using the B-cell isolation kit II according to the instructions of the manufacturer (Miltenyi Biotech).

Mordasini V., Ueda S., Aslandogmus R., Berger C., Gysin C., Hühn D., Sartori A.A., Bernasconi M, & Nadal D. (2016). Activation of ATR-Chk1 pathway facilitates EBV-mediated transformation of primary tonsillar B-cells. Oncotarget, 8(4), 6461-6474.

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Isolation of Peripheral Blood Immune Cells

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Peripheral blood samples were obtained from healthy donors after informed consent and approval was obtained by the ethical committee of the hospital according to the Helsinki declaration. PBMCs were isolated using Ficoll gradient (Sigma-Aldrich, St Louis, MO). CD19⁺ B cells and CD3⁺ T cells were purified by negative selection using the B cell isolation Kit II (Miltenyi Biotec) for CD19⁺ B cells and the human Pan T cells isolation kit (Miltenyi Biotec) for the CD3⁺ T cells following manufacturer's instructions followed by separation through the Automacs pro-separator (Miltenyi Biotec). Purity of isolated B and T cells was monitored using anti-hCD19APC and anti-hCD3PE antibodies (Miltenyi Biotec), respectively, and was analyzed by flow cytometry (MACSQuant VYB, Milteny Biotech).

Gutierrez-Guerrero A., Abrey Recalde M.J., Mangeot P.E., Costa C., Bernadin O., Périan S., Fusil F., Froment G., Martinez-Turtos A., Krug A., Martin F., Benabdellah K., Ricci E.P., Giovannozzi S., Gijsbers R., Ayuso E., Cosset F.L, & Verhoeyen E. (2021). Baboon Envelope Pseudotyped “Nanoblades” Carrying Cas9/gRNA Complexes Allow Efficient Genome Editing in Human T, B, and CD34+ Cells and Knock-in of AAV6-Encoded Donor DNA in CD34+ Cells. Frontiers in Genome Editing, 3, 604371.

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Isolation and Co-culture of pDCs and B Cells

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Peripheral blood mononuclear cells (PBMCs) were purified from healthy blood donor buffy coats or EDTA-blood from SLE patients and healthy donors using Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden) density-gradient centrifugation. PDCs and B cells were isolated from PBMCs by magnetic bead separation using negative selection (pDC Isolation kit and B cell Isolation kit II; Miltenyi Biotec, Bergisch Gladbach, Germany). Purity of the isolated cell populations was determined by flow cytometry after staining with anti-BDCA2 (Miltenyi Biotec) or anti-CD19 (BD Biosciences) monoclonal antibodies (mAbs) and was found to be at least 95%. The pDCs and B cells were cultured as previously described with 0.25x10⁵ pDCs and 1x10⁵ B cells in 0.1 ml volumes per well in 96-well plates for 20 hours or 4 to 6 days as indicated, at 37°C with 5% CO₂ [17 (link)]. When indicated, affinity purified polyclonal goat anti-BAFF (#AF124) or normal goat IgG antibodies (R&D Systems) were added to the co-cultures of pDCs and B cells at a final concentration of 20 μg/ml at the beginning of the cell culturing.

Berggren O., Hagberg N., Alexsson A., Weber G., Rönnblom L, & Eloranta M.L. (2017). Plasmacytoid dendritic cells and RNA-containing immune complexes drive expansion of peripheral B cell subsets with an SLE-like phenotype. PLoS ONE, 12(8), e0183946.

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Isolation and Culture of Primary T and B Cells

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Human Peripheral blood was used to isolate peripheral blood mononuclear cells (PBMC) by density centrifugation method according to the manufacturer’s guideline (Sigma, San Louis, MO, USA). Primary T cells were positively selected using a mixture of (1:1) anti-CD4 and anti-CD8 microbeads (Miltenyi, Koln, Germany) and normal B cells were negative selected by using B Cell Isolation Kit II (Miltenyi) from PBMCs according to the manufacturer’s protocol. Both of them were cultured in X-VIVO^TM 15 serum free medium (LONZA, Basel, Switzerland) supplemented with 1% P/S, and stored in liquid nitrogen. All fresh blood was collected under a protocol approved by the Ethics Committee of East China Normal University (m20190315), following written informed consent.

Tao L., Farooq M.A., Gao Y., Zhang L., Niu C., Ajmal I., Zhou Y., He C., Zhao G., Yao J., Liu M, & Jiang W. (2020). CD19-CAR-T Cells Bearing a KIR/PD-1-Based Inhibitory CAR Eradicate CD19+HLA-C1− Malignant B Cells While Sparing CD19+HLA-C1+ Healthy B Cells. Cancers, 12(9), 2612.

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Single-cell Sorting of HIV-1 Env-Reactive B Cells

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B cells were isolated from PBMCs using the Pan B Cell Isolation Kit, B Cell Isolation Kit II, or IgG⁺ Memory B Cell Isolation Kit (Miltenyi Biotec). Isolated cells were labeled with anti-human CD19-AF700 (BD), anti-human IgG-APC (BD), DAPI (Thermo Fisher), and the respective HIV-1 Env bait for 30 minutes on ice. BG505_SOSIP.664-GFP or biotinylated YU2_gp140 that was labeled with Streptavidin-PE (BD) were used as HIV-1 Env baits. Env-reactive CD19⁺IgG⁺DAPI^- single cells were sorted into 96-well plates containing 4 μl of lysis buffer (0.5x PBS, 10 mM DTT (Thermo Fisher), 2 U/μl RNasin (Promega), and 1 U/μl RNaseOUT (Thermo Fisher)) per well using a BD FACSAria Fusion. Plates were stored at −80°C until further use.

Schommers P., Gruell H., Abernathy M.E., Tran M.K., Dingens A.S., Gristick H.B., Barnes C.O., Schoofs T., Schlotz M., Vanshylla K., Kreer C., Weiland D., Holtick U., Scheid C., Valter M.M., van Gils M.J., Sanders R.W., Vehreschild J.J., Cornely O.A., Lehmann C., Fätkenheuer G., Seaman M.S., Bloom J.D., Bjorkman P.J, & Klein F. (2020). Restriction of HIV-1 Escape by a Highly Broad and Potent Neutralizing Antibody. Cell, 180(3), 471-489.e22.

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B-cell Differentiation Kinetics

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Peripheral blood mononuclear cells (PBMC) were ordered and B cells were isolated using the Magnetic-activated cell sorting (MACS), specifically the human Miltenyi B-cell isolation kit II. Cells were then seeded. Controls were stimulated with LPS while non-controls were stimulated using one of the following stimulation cocktails: IgM.acD40.IL4.IL21, TGFb.acD40.IL4. Cells were harvested at days 1, 3 and 9 following seeding. The time course experiment was performed twice in parallel resulting in two replicates for every condition at all three time points.

Lefol Y., Korfage T., Mjelle R., Prebensen C., Lüders T., Müller B., Krokan H., Sarno A., Alsøe L., Berdal J.E., Sætrom P., Nilsen H, & Domanska D. (2023). TiSA: TimeSeriesAnalysis—a pipeline for the analysis of longitudinal transcriptomics data. NAR Genomics and Bioinformatics, 5(1), lqad020.

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Single-cell analysis of B-cell memory

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PBMCs from D7 and D9, which correspond to the peak of the B cell memory recall response, were thawed. B cells were magnetically enriched using the B Cell Isolation Kit II (Miltenyi). Single cells were encapsulated in droplets using 16 lanes of the Chromium device (10X Genomics) with target loading of 14,000 cells per lane. Reverse transcription and complementary DNA (cDNA) amplification were performed using the Single Cell V(D)J kit (10X Genomics). In 12 lanes, direct enrichment of VDJ was performed. In the remaining 4 lanes, VDJ and gene expression measurement was performed; these 4 lanes were considered technical replicates. All steps were done according to manufacturer’s instructions, except with additional cycles of polymerase chain reaction (PCR) (19 total cycles for direct enrichment of VDJ; 22 total cycles for VDJ and gene expression). 50 ng of cDNA was used as input for library preparation. Libraries were sequenced using the Illumina NextSeq 500 platform with paired-end reads for VDJ of 150 bp each and for gene expression of 26 bp and 98 bp.

Horns F., Dekker C.L, & Quake S.R. (2020). Memory B Cell Activation, Broad Anti-influenza Antibodies, and Bystander Activation Revealed by Single-Cell Transcriptomics. Cell reports, 30(3), 905-913.e6.

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PBMC Isolation and B-Cell Enrichment

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Heparinized peripheral blood (18 mL) was collected, of which 3 mL was set aside for stimulation. From the remaining whole blood, PBMCs were isolated using the ficoll‐histopaque (GE Healthcare Life Sciences, Marlborough, MA) density gradient method. A fraction of the PBMCs (3 × 10⁶ cells) was set aside for stimulation. Subsequently, B‐cells were negatively isolated from the remaining PBMCs using the magnetic‐activated cell sorting bead technology, according to the manufacturer's instructions, with the B‐Cell Isolation Kit II (Miltenyi Biotec, Centurion, South Africa). Once all the sample fractions had been collected, the cells were stimulated as described below. The purity of the enriched B‐cell samples was confirmed by flow cytometry using anti‐human CD19 mAb. All samples with resulting gated purity of above 90% were included in analysis.

Moore D.K., Leisching G.R., Snyders C.I., Gutschmidt A., van Rensburg I.C, & Loxton A.G. (2020). Immunoglobulin profile and B‐cell frequencies are altered with changes in the cellular microenvironment independent of the stimulation conditions. Immunity, Inflammation and Disease, 8(3), 458-467.

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Single-cell analysis of B-cell memory

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Isolation and Purification of PBMC Subsets

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Peripheral blood mononuclear cells (PBMCs) were separated immediately after blood sample collection, by Ficoll-Hypaque (GE Healthcare Biosciences AB, Uppsala, Sweden) density gradient centrifugation. CD4+ T or B cells were purifed from freshly isolated PBMCs by exhaustive immunomagnetic negative selection in an Automacs (Miltenyi Biotec, Bergisch Gladbach, Germany), using the “CD4+ T Cell Isolation Kit” or the “B Cell Isolation Kit II” from Miltenyi Biotec. Isolated CD4+ T cells or CD19+ B cells were >98% pure. CXCR5+ and CXCR5- subpopulations were subsequently isolated from total CD4+ T cells using PE-labeled CXCR5 microbeads (Miltenyi Biotec). Naïve (CD19+CD27-) and memory (CD19+CD27+) B cells were selected from total CD19+ B cells using CD27+ microbeads (Miltenyi Biotec). T and B cell subpopulations were >98% pure and used immediately after isolation.

Bautista-Caro M.B., Arroyo-Villa I., Castillo-Gallego C., de Miguel E., Peiteado D., Plasencia-Rodríguez C., Villalba A., Sánchez-Mateos P., Puig-Kröger A., Martín-Mola E, & Miranda-Carús M.E. (2014). Decreased Frequencies of Circulating Follicular Helper T Cell Counterparts and Plasmablasts in Ankylosing Spondylitis Patients Naïve for TNF Blockers. PLoS ONE, 9(9), e107086.

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