Easily and efficiently isolate highly purified human B cells, including plasma cells, from fresh or previously frozen human peripheral blood mononuclear cells (PBMCs) or lysed leukapheresis samples by immunomagnetic negative selection, with the EasySep™ Human Pan-B Cell Enrichment Kit. Widely used in published research for more than 20 years, EasySep™ combines the specificity of monoclonal antibodies with the simplicity of a column-free magnetic system.
In this EasySep™ negative selection procedure, unwanted cells are labeled with antibody complexes and magnetic particles. Unwanted cells expressing the following markers are targeted for removal: CD2, CD3, CD14, CD16, CD36, CD42b, CD56, CD66b, CD123, and glyA. The magnetically labeled cells are then separated from the untouched desired B cells by using an EasySep™ magnet and simply pouring or pipetting the desired cells into a new tube. Following magnetic cell isolation, the desired B cells are ready for downstream applications such as flow cytometry, culture, or DNA/RNA extraction.
For isolation of only CD43-negative B cells from normal samples, we recommend using EasySep™ Human B Cell Enrichment Kit (Catalog #19054).
For enrichment of B cells from peripheral blood or other tissues of patients with B cell leukemia or lymphoma, or with other disease states in which B cells may express CD43, CD36, and/or CD123, we recommend using EasySep™ Human B Cell Enrichment Kit II Without CD43 Depletion (Catalog #17963).
Learn more about how immunomagnetic EasySep™ technology works or how to fully automate immunomagnetic cell isolation with RoboSep™. Explore additional products optimized for your workflow, including culture media, supplements, antibodies, and more.
In this EasySep™ negative selection procedure, unwanted cells are labeled with antibody complexes and magnetic particles. Unwanted cells expressing the following markers are targeted for removal: CD2, CD3, CD14, CD16, CD36, CD42b, CD56, CD66b, CD123, and glyA. The magnetically labeled cells are then separated from the untouched desired B cells by using an EasySep™ magnet and simply pouring or pipetting the desired cells into a new tube. Following magnetic cell isolation, the desired B cells are ready for downstream applications such as flow cytometry, culture, or DNA/RNA extraction.
For isolation of only CD43-negative B cells from normal samples, we recommend using EasySep™ Human B Cell Enrichment Kit (Catalog #19054).
For enrichment of B cells from peripheral blood or other tissues of patients with B cell leukemia or lymphoma, or with other disease states in which B cells may express CD43, CD36, and/or CD123, we recommend using EasySep™ Human B Cell Enrichment Kit II Without CD43 Depletion (Catalog #17963).
Learn more about how immunomagnetic EasySep™ technology works or how to fully automate immunomagnetic cell isolation with RoboSep™. Explore additional products optimized for your workflow, including culture media, supplements, antibodies, and more.
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Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.
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Resources and Publications
Educational Materials (9)
Frequently Asked Questions
Publications (5)
FLAIRR-Seq: A Method for Single-Molecule Resolution of Near Full-Length Antibody H Chain Repertoires. Journal of immunology (Baltimore, Md. : 1950) 2023 may
Abstract
Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article, we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE, combined with single-molecule, real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV), D (IGHD), and J (IGHJ) gene usage, complementarity-determining region 3 length, and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together, these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide, for the first time, to our knowledge, simultaneous single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date.
Germinal centre-driven maturation of B cell response to mRNA vaccination. Nature 2022 apr
Abstract
Germinal centres (GC) are lymphoid structures in which B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells1-5 (BMPCs). SARS-CoV-2 mRNA vaccination induces a persistent GC response that lasts for at least six months in humans6-8. The fate of responding GC B cells as well as the functional consequences of such persistence remain unknown. Here, we detected SARS-CoV-2 spike protein-specific MBCs in 42 individuals who had received two doses of the SARS-CoV-2 mRNA vaccine BNT162b2 six month earlier. Spike-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of sequencing the B cell receptors of responding blood plasmablasts and MBCs, lymph node GC B cells and plasma cells and BMPCs from eight individuals and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1,540 spike-specific B cell clones. On average, early blood spike-specific plasmablasts exhibited the lowest SHM frequencies. By contrast, SHM frequencies of spike-specific GC B cells increased by 3.5-fold within six months after vaccination. Spike-specific MBCs and BMPCs accumulated high levels of SHM, which corresponded with enhanced anti-spike antibody avidity in blood and enhanced affinity as well as neutralization capacity of BMPC-derived monoclonal antibodies. We report how the notable persistence of the GC reaction induced by SARS-CoV-2 mRNA vaccination in humans culminates in affinity-matured long-term antibody responses that potently neutralize the virus.
B-1b Cells Possess Unique bHLH-Driven P62-Dependent Self-Renewal and Atheroprotection. Circulation research 2022 apr
Abstract
BACKGROUND B1a and B1b lymphocytes produce IgM that inactivates oxidation-specific epitopes (IgMOSE) on LDL (low-density lipoprotein) and protects against atherosclerosis. Loss of ID3 (inhibitor of differentiation 3) in B cells selectively promotes B1b but not B1a cell numbers, leading to higher IgMOSE production and reduction in atherosclerotic plaque formation. Yet, the mechanism underlying this regulation remains unexplored. METHODS Bulk RNA sequencing was utilized to identify differentially expressed genes in B1a and B1b cells from Id3KO and Id3WT mice. CRISPR/Cas9 and lentiviral genome editing coupled with adoptive transfer were used to identify key Id3-dependent signaling pathways regulating B1b cell proliferation and the impact on atherosclerosis. Biospecimens from humans with advanced coronary artery disease imaging were analyzed to translate murine findings to human subjects with coronary artery disease. RESULTS Through RNA sequencing, P62 was found to be enriched in Id3KO B1b cells. Further in vitro characterization reveals a novel role for P62 in mediating BAFF (B-cell activating factor)-induced B1b cell proliferation through interacting with TRAF6 (tumor necrosis factor receptor 6) and activating NF-$\kappa$B (nuclear factor kappa B), leading to subsequent C-MYC (C-myelocytomatosis) upregulation. Promoter-reporter assays reveal that Id3 inhibits the E2A protein from activating the P62 promoter. Mice adoptively transferred with B1 cells overexpressing P62 exhibited an increase in B1b cell number and IgMOSE levels and were protected against atherosclerosis. Consistent with murine mechanistic findings, P62 expression in human B1 cells was significantly higher in subjects harboring a function-impairing single nucleotide polymorphism (SNP) at rs11574 position in the ID3 gene and directly correlated with plasma IgMOSE levels. CONCLUSIONS This study unveils a novel role for P62 in driving BAFF-induced B1b cell proliferation and IgMOSE production to attenuate diet-induced atherosclerosis. Results identify a direct role for Id3 in antagonizing E2A from activating the p62 promoter. Moreover, analysis of putative human B1 cells also implicates these pathways in coronary artery disease subjects, suggesting P62 as a new immunomodulatory target for treating atherosclerosis.
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