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Easysep human memory b cell isolation kit

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The EasySep Human Memory B Cell Isolation Kit is a lab equipment product designed to isolate memory B cells from human peripheral blood mononuclear cells (PBMCs). It utilizes a magnetic separation technique to selectively enrich for the desired cell population.

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Lab products found in correlation

Easysep human pan b cell enrichment kit, by STEMCELL (2 mentions) Easysep human na ve b cell enrichment kit, by STEMCELL (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) Cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions) L glutamate, by Thermo Fisher Scientific (1 mentions) Anti cd3 cd28, by BD (1 mentions) Class b cpg oligodeoxynucleotides, by InvivoGen (1 mentions) Histopaque 1077, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Corning (1 mentions) Chromium single cell 5 d j kit, by 10x Genomics (1 mentions) Novaseq platform, by Illumina (1 mentions)

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EasySep Human B Cell Isolation Kit EasySep B cell isolation kit EasySep cell isolation kit EasySep isolation kits Cell isolation kits EasySep kits Isolation kits

8 protocols using easysep human memory b cell isolation kit

1

Isolation of Memory and Naïve B Cells

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To isolate human memory and naïve B cells from 14 healthy individuals and 18 HCV patients, EasySep Human Memory B Cell Isolation Kit and EasySep Human Naïve B Cell Enrichment Kit (STEMCELL Technologies, Co. Ltd, USA) were used, respectively according to instruction manuals. IRB approval is obtained by University committee. Exosome isolation is described in Supplementary Methods.
Chen C.L., Huang J.Y., Wang C.H., Tahara S.M., Zhou L., Kondo Y., Schechter J., Su L., Lai M.M., Wakita T., Cosset F.L., Jung J.U, & Machida K. (2017). Hepatitis C virus has a genetically determined lymphotropism through co-receptor B7.2. Nature Communications, 8, 13882.
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2

Memory B cell function with CD8+ T cells

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CD19+ CD27+ memory B (MB) cells were isolated using the EasySep Human Memory B Cell Isolation Kit (STEMCELL Technologies, Canada) according to the manufacturer’s instructions. CD8+T cells were isolated using a CD8+ T Cell Isolation Kit (Miltenyi, Germany). MB cells (1×105 /mL) from SLE and Healthy donors were cultured alone or in the presence of autologous CD8+ T cells (4×105 /mL) at 1:4 ratio for 3 days at 37°C. Co-cultured cells were incubated in complete RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen) supplemented with 25 mM HEPES (Invitrogen), 2 mM L-glutamate (Invitrogen) and 1% non-essential amino acids (Life Technologies). During incubation, cells were stimulated with class B CpG oligodeoxynucleotides (InvivoGen) at 5 µg/mL, 1 µg/mL anti-CD3/CD28 (BD Biosciences, USA).
Wang K., Zhao J., Feng X., He S., Li J., Sun F., Xu Z., Yang H., Ye J., Cao L, & Ye S. (2024). PD-1/PD-L1 governed cross-talk of exhausted CD8+ T and memory B cells in systemic lupus erythematosus. RMD Open, 10(1), e003503.
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3

Isolation and Characterization of Primary Human B Cells

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Adult buffy coats were obtained from the Gulf Coast Regional Blood Center (Pro00006262) and cord blood was obtained from the Carolinas Cord Blood Bank (Pro00061264). Peripheral blood mononuclear cells were isolated using Histopaque-1077 (Sigma, H8889) and SepMate PBMC Isolation tubes. Naïve B cells were then isolated from adult blood using the EasySep Human Memory B Cell Isolation kit (STEMCELL Technologies #17864) to first remove CD27 positive memory cells, followed by negative isolation of B cells. To obtain total B cells, the Easy Human Pan-B Cell Enrichment kit (STEMCELL Technologies #19554) was used. Purity of isolated cells was assessed by flow cytometry with antibodies to CD19, IgD, and CD27 (Table 1). Primary cells were maintained in Roswell Park Memorial Institute (RPMI) medium 1640 supplemented with 20% heat-inactivated fetal bovine serum (FBS) (Corning). LCLs and BJAB cell lines were maintained in RPMI supplemented with 10% FBS and 2 mM L-glutamine, penicillin/streptomycin. 293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS.
Cable J.M., Reinoso-Vizcaino N.M., White R.E, & Luftig M.A. (2024). Epstein-Barr virus protein EBNA-LP engages YY1 through leucine-rich motifs to promote naïve B cell transformation. bioRxiv.
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4

Single-cell Ig-Seq analysis of CSF and PBMC

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CSF and blood were obtained during diagnostic and research procedures after subjects had provided informed consent; 20 to 30 mL of CSF was centrifuged immediately after collection at 400 × g for 15 min at 4 °C. The pellet was resuspended in ∼80 µL of residual supernatant, and lymphocytes were counted using a hemocytometer. Peripheral blood mononuclear cells (PBMCs) were isolated using cell preparation tubes as described previously (17 ) and resuspended in 2% fetal bovine serum. All experiments were performed immediately with freshly collected, unsorted cells. To obtain additional single-cell Ig-Seq (scIg-Seq) data for some patients, CSF or PBMCs were enriched for total B cells or for memory B cells via bead-based selection (EasySep Human Pan-B cell enrichment kit and EasySep Human Memory B cell Isolation Kit, respectively; StemCell Technologies). Sequencing libraries were prepared using 3′ or 5′ library preparation kits (10x Genomics).
Ramesh A., Schubert R.D., Greenfield A.L., Dandekar R., Loudermilk R., Sabatino JJ J.r., Koelzer M.T., Tran E.B., Koshal K., Kim K., Pröbstel A.K., Banerji D., Guo C.Y., Green A.J., Bove R.M., DeRisi J.L., Gelfand J.M., Cree B.A., Zamvil S.S., Baranzini S.E., Hauser S.L, & Wilson M.R. (2020). A pathogenic and clonally expanded B cell transcriptome in active multiple sclerosis. Proceedings of the National Academy of Sciences of the United States of America, 117(37), 22932-22943.
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5

Isolation and Co-culture of T Follicular Helper-like Cells and Memory B Cells

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After 4 days of co-culture with total GM-CSF-DCs, CD4+ T cells were FACS sorted as PD-1High CXCR5+ (Tfh-like cells) or PD-1Low CXCR5+ (TLow cells). Allogeneic PBMCs were thawed and, after a round of human memory B cell enrichment, memory B cells were magnetically sorted using the EasySep Human Memory B cell isolation kit (StemCell Technologies). T cells and B cells were co-cultured in X-VIVO medium in round-bottom plates (2.5×105 T cells and 2.5×105 memory B cells). At day 10 of culture, cells were harvested for FACS analysis.
Korniotis S., Saichi M., Trichot C., Hoffmann C., Amblard E., Viguier A., Grondin S., Noel F., Mattoo H, & Soumelis V. (2022). GM-CSF-activated human dendritic cells promote type 1 T follicular helper cell polarization in a CD40-dependent manner. Journal of Cell Science, 135(21), jcs260298.
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6

Isolation and Characterization of SARS-CoV-2 Neutralizing Antibody

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CoV11 was isolated from a 76-year-old man with history of severe COVID-19, infected in February 2020 with high titer of neutralizing antibodies against the virus. CoV11 (also known as CoVIC-079) has been previously published (22 (link)). Briefly, memory B cells were isolated from PBMCs using the EasySep™ Human Memory B Cell Isolation Kit (STEMCELL Technologies). Cells were incubated with biotin-conjugated SARS-CoV-2 Spike antigen (LakePharma) and then, after washing, labeled by incubation with TotalSeq™-C0953 PE Streptavidin (BioLegend). The cell surface labeled single-cell suspension was loaded onto a 10x Genomics Chromium Controller microfluidics chip (10x Genomics) and a VDJ library was prepared based on the manufacturer’s instructions. A subset of cells containing the surface barcode were selected and their VDJ sequences were cloned into IgG1 heavy and light chain vectors. The recombinant plasmids were then co-transfected into FreeStyle-293 cells for expression and the secreted antibodies were purified from culture supernatants after incubations of 1 week by Protein A affinity chromatography. CoV11 was identified as one of the antibodies that strongly neutralized the virus.
Tolbert W.D., Chen Y., Sun L., Benlarbi M., Ding S., Manickam R., Pangaro E., Nguyen D.N., Gottumukkala S., Côté M., Gonzalez F.J., Finzi A., Tehrani Z.R., Sajadi M.M, & Pazgier M. (2023). The molecular basis of the neutralization breadth of the RBD-specific antibody CoV11. Frontiers in Immunology, 14, 1178355.
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7

Comprehensive Single-Cell Analysis of B Cells

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CSF supernatant was removed, the cell pellet was gently resuspended, and lymphocytes were counted via hemocytometer. An aliquot of PBMCs was enriched for B cells, and a second aliquot was enriched for memory B cells via bead-based selections (EasySep Human Pan-B cell enrichment kit, and EasySep Human Memory B Cell Isolation Kit, respectively, StemCell Technologies). Isolated peripheral blood B cells and unenriched PBMCs were resuspended in 2% serum. Single cells were isolated on a droplet-based single cell isolation platform (10X Genomics), and individual heavy and light chain Ig sequences were identified using the Chromium Single Cell V(D)J kit (10X Genomics) and paired-end 150bp × 150 bp sequencing on an Illumina NovaSeq platform. Two lanes of CSF cells were isolated as single cells on the same platform: 7,711 and 4,938 cell input, respectively, one lane of 17,400 PBMCs, one lane of 18,125 peripheral blood B cells, and one lane of 18,565 peripheral blood memory B cells. Estimated cell count input was based on manual hemocytometer count.
Pröbstel A.K., Zhou X., Baumann R., Wischnewski S., Kutza M., Rojas O.L., Sellrie K., Bischof A., Kim K., Ramesh A., Dandekar R., Greenfield A.L., Schubert R.D., Bisanz J.E., Vistnes S., Khaleghi K., Landefeld J., Kirkish G., Liesche-Starnecker F., Ramaglia V., Singh S., Tran E.B., Barba P., Zorn K., Oechtering J., Forsberg K., Shiow L.R., Henry R.G., Graves J., Cree B.A., Hauser S.L., Kuhle J., Gelfand J.M., Andersen P.M., Schlegel J., Turnbaugh P.J., Seeberger P.H., Gommerman J.L., Wilson M.R., Schirmer L, & Baranzini S.E. (2020). Gut microbiota-specific IgA+ B cells traffic to the CNS in active multiple sclerosis. Science immunology, 5(53), eabc7191.
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8

Isolation of CD27+ Memory B Cells

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CD27+ memory B cells were isolated from purified B cells by immunomagnetic positive selection according to the manufacturer’s protocol (EasySep Human Memory B Cell Isolation Kit, STEMCELL). Briefly, CD27+ B cells are labeled with magnetic beads combined with CD27 antibodies and separated using an EasySep magnet. Purified CD27+ B cells were eluted and washed in PBS containing 2% (v/v) fetal bovine serum (FBS) and 1 mM EDTA. CD27+ B cells were counted by using 0.4% (w/v) trypan blue stain and Countess Automated Cell Counter according to the manufacturer’s protocol.
Cao Y., Su B., Guo X., Sun W., Deng Y., Bao L., Zhu Q., Zhang X., Zheng Y., Geng C., Chai X., He R., Li X., Lv Q., Zhu H., Deng W., Xu Y., Wang Y., Qiao L., Tan Y., Song L., Wang G., Du X., Gao N., Liu J., Xiao J., Su X.D., Du Z., Feng Y., Qin C., Qin C., Jin R, & Xie X.S. (2020). Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients’ B Cells. Cell, 182(1), 73-84.e16.
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