Easysep b cell isolation kit

Manufactured by STEMCELL

Sourced in United States

The EasySep B cell isolation kit is a laboratory tool used for the separation and isolation of B cells from a mixed cell population. The kit utilizes magnetic particles and customized cocktails of antibodies to selectively target and remove non-B cells, allowing for the enrichment of the desired B cell population.

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Lab products found in correlation

Fluo 4 am, by Thermo Fisher Scientific (4 mentions) Fluo 4, by Tecan (2 mentions) Malachite green assay, by R&D Systems (1 mentions) Pom 1, by Thermo Fisher Scientific (1 mentions) Counting beads, by Thermo Fisher Scientific (1 mentions) Easy sep cd4 isolation kit, by STEMCELL (1 mentions) Cd45ro pe cy7 clone uchl1, by BD (1 mentions) Cd8 apc clone rpa t8, by BD (1 mentions) Cd4 bv605 clone rpa t4, by BD (1 mentions) Cxcr5 bv421 clone j252d4, by BioLegend (1 mentions) L d aqua, by Thermo Fisher Scientific (1 mentions) Benzonase nuclease, by Merck Group (1 mentions) Nc 3000, by ChemoMetec (1 mentions) Fcr block, by BioLegend (1 mentions) Superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions) Efluor 780, by Thermo Fisher Scientific (1 mentions) Ficoll paque plus, by GE Healthcare (1 mentions) Easysep cd8 t cell isolation kit, by STEMCELL (1 mentions) Nhp cd14 positive selection kit, by Miltenyi Biotec (1 mentions) Fc block, by BioLegend (1 mentions)

Close spelling variants of this product

EasySep T cell or B cell Isolation Kits EasySep Human Naïve B Cell Isolation Kit EasySep Human Memory B Cell Isolation Kit EasySep Mouse Pan-B cell Isolation Kit EasySep™ Human Naive B Cell Isolation Kit EasySep Direct Human B Cell Isolation Kit EasySep Mouse B Cell Isolation Kit EasySep Human B Cell Isolation Kit EasySep cell isolation kit EasySep isolation kits

12 protocols using easysep b cell isolation kit

Inhibition of CD39 on B Cells

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Example 10

B cells were isolated from human leukopak using EasySep B cell isolation kit (STEMCELL Technologies). 5×10⁴B cells/well were washed with Tris buffer and incubated with serially diluted (100-0.00013 nM) antibodies for 30 minutes at 37 degrees C. 50 μM ATP was added to each well and incubated with cells for 2 hrs. The supernatants were collected and analyzed in Malachite Green Assay (R&D) according to manufacturer's protocol. Phosphate released from CD39 processing of ATP was used as a readout of enzyme activity. Palivizumab was used as an isotype control and ARL (Tocris) and POM-1 (Alpha Aesar), non-specific small molecule inhibitors of CD39, were used as positive controls at 100 μM.

The antibodies were demonstrated to bind to primary human and cyno B cells (see FIG. 7) and the next step was to evaluate the inhibition of ATP hydrolysis by detection of free phosphate (Pi) using a Malachite Green Assay. The results are shown in FIG. 8 and indicate the anti-CD39 antibodies inhibit the enzymatic inhibition/dephosphorylation of ATP by primary human B cells. The ability of the antibodies to inhibit enzymatic activity was comparable regardless of high vs. low max MFI detected in the binding to human B cells (FIG. 7).

US11345757B2. Anti-CD39 antibodies (2022-05-31). Trishula Therapeutics, Inc. [US]. Inventors: Vanessa Soros [US], Maria Kovalenko [US], John Corbin [US], Courtney Beers [US], Paul Fredrick Widboom [US], Joseph Robert Warfield [US].

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Ramos B Cell Calcium Flux Assay

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Ramos B Cells at a concentration of 10 million cells/mL were incubated with 10 μM Fluo-4 AM (ThermoFisher, Inc.) for 30 minutes at 37C. After washing once, flux assays were performed on a Tecan plate reader at 37C on a 96 well microplate with 160 μL of Fluo-4 labeled Ramos cells at 2 million cells/mL. A baseline fluorescence was then recorded for 1 minute, and 40 μL of NPs were added to the cells for a final concentration of 5 nM of antigen, unless otherwise stated. A fixed concentration of antigens was used rather than the concentration of DNA-NPs to simplify the comparison between experiments with various DNA-NPs and to assess the role of antigen concentration instead of the role of the DNA-NPs. For studies utilizing the p5 peptide antigen, primary B cells were isolated from 3–83 mouse spleens and stained via the same procedure. Primary B cells were isolated from splenocytes by negative selection using a StemCell EasySep B Cell Isolation Kit.

Veneziano R., Moyer T.J., Stone M.B., Wamhoff E.C., Read B.J., Mukherjee S., Shepherd T.R., Das J., Schief W.R., Irvine D.J, & Bathe M. (2020). Role of nanoscale antigen organization on B-cell activation probed using DNA origami. Nature nanotechnology, 15(8), 716-723.

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Quantifying B-cell Differentiation Induced by Antigen-specific CD4 T Cells

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PBMC were thawed in complete 10% FBS RPMI media (Millipore Sigma) with Benzonase® Nuclease (Millipore Sigma). CD4 T cells were isolated by magnetic bead negative selection with the EasySep CD4 isolation kit (STEMCell Technologies). Cells were stained for L/D-AQUA (Thermo Fisher) and then extracellular staining was done using: CD4-BV605 (clone RPA-T4, BD Bioscience), CD45RO-PE-Cy7 (clone UCHL1, BD Biosciences), CD8-APC (clone RPA-T8, BD), CXCR5-BV421(clone J252D4, Biolegend), and CD3-PE (SK7, Biolegend). Stained cells were sorted on a BD FACSAria™. 50,000 sorted CD45RO⁺CXCR5⁺ or CD45RO⁺CXCR5^- cells were then plated in a 96 U bottom plate. B cells from a non-related healthy control donor were isolated by magnetic bead negative selection with the EasySep B cell isolation kit (STEMCell Technologies). 50,000 B cells were added to the corresponding 96 U bottom plate in 10% FBS RPMI media (Millipore Sigma) with antiretroviral drugs were added to the culture (200nM raltegravir, 200nM lamivudine) (NIH AIDS reagent program). After 7 days of co-culturing in a 37°C incubator, B-cell differentiation was determined by flow cytometry and absolute cell numbers were quantified using counting beads (Thermo Fisher). Staining for B-cell differentiation was performed using antibodies listed in Supplementary Table 3, panel 5.

Wong C.S., Buckner C.M., Lage S.L., Pei L., Assis F.L., Dahlstrom E.W., Anzick S.L., Virtaneva K., Rupert A., Davis J.L., Zhou T., Laidlaw E., Manion M., Galindo F., Anderson M., Seamon C.A., Sneller M.C., Lisco A., Deleage C., Pittaluga S., Moir S, & Sereti I. (2021). Rapid Emergence of T Follicular Helper and Germinal Center B Cells Following Antiretroviral Therapy in Advanced HIV Disease. Frontiers in Immunology, 12, 752782.

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Competitive Transfer of Labeled B Cells

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For splenic B cell cultures, B cells were isolated using the EasySep B cell isolation kit (Stemcell) and cultured for 3 days in R10 medium supplemented with isotype switching cytokines (isotype switch mix, see above). Three days after stimulation, cell cultures of WT cre+ and KLF2-deficient B cells were analyzed for viability, activation and class-switching by flow cytometry (Fig. S4G). 8 × 10⁶ living cells per culture were harvested (cell counts and viabilities were determined by NC3000, Chemometec) and reciprocally labeled with either eFluor450 or eFluor670 proliferation dyes. Setting 1: eFluor450-labeled WT cre+ cells were equally mixed with eFluor670-labeled KLF2 KO cells. Setting 2: eFluor670-labeled WT cre+ cells were equally mixed with eFluor450-labeled KLF2 KO cells. For competitive transfer, cell mixtures of 16 × 10⁶ cells were resuspended in 75 µl sterile PBS and retroorbitally i.v injected into Rag^−/− recipient mice. Spleen, BM, mLN, blood and cLP of Rag^−/− recipient mice were analyzed by flow cytometry for the presence of transferred, labeled cells 3 days after i.v. injection to identify injected donor PB/PC within the recipient mice.

Wittner J., Schulz S.R., Steinmetz T.D., Berges J., Hauke M., Channell W.M., Cunningham A.F., Hauser A.E., Hutloff A., Mielenz D., Jäck H.M, & Schuh W. (2022). Krüppel-like factor 2 controls IgA plasma cell compartmentalization and IgA responses. Mucosal Immunology, 15(4), 668-682.

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Isolation and Imaging of Human Tonsil B Cells

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Mononuclear cells isolated from tonsil tissue were treated with FcR block (Biolegend—Cat: 422302) before staining with fluorescent antibodies: anti-CD19 (HIB19), anti-IgM (MHM-88), anti-IgD (IA6-2), anti-CD27 (LG.3A10). Dead cells were excluded with fixable cell viability dye (eBioscience—eFluor 780). Naïve and memory B cells were analyzed and sorted using FACSAria III. Negative selection of B cells was performed with StemCell—EasySep B cell isolation kit (Cat: 17954) and shown by flow cytometry to be >98% pure. B cells were counted and plated on glass slides (Fisherbrand—Superfrost Plus microscope slides) or glass coverslips at high density (≥5 × 10⁴ cell/cm²) or low density (5 × 10³ cell/cm²). The cells were cultured in FCS to facilitate imaging and were incubated in humidified chambers at 37 °C and 5% CO₂ for 1–3 h. The cells were fixed with 100% methanol or 4% paraformaldehyde and stained with Wright- Giemsa or immunofluorescence for analysis by microscopy.

Bukhari A., Kalinina O, & Knight K.L. (2023). Death of tonsillar B cells by NETosis. Cell Death Discovery, 9, 108.

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Isolation of Primary Human B-Cells

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Primary human B-cells were isolated from whole blood using Ficoll-Paque PLUS (GE Healthcare) as previously described [97 (link)]. B cells were then isolated by negative selection using EasySep B-cell Isolation Kit (StemCell Technologies) according to the manufacturer’s instructions.

Hunte R., Alonso P., Thomas R., Bazile C.A., Ramos J.C., van der Weyden L., Dominguez-Bendala J., Khan W.N, & Shembade N. (2018). CADM1 is essential for KSHV-encoded vGPCR-and vFLIP-mediated chronic NF-κB activation. PLoS Pathogens, 14(4), e1006968.

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Isolation and Purification of Naive and Memory CD8+ T Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll-Hypaque density gradient and either used immediately or stored in liquid nitrogen. CD8⁺ T cells from normal PBMCs were negatively selected using EasySep CD8⁺ T-cell isolation kit (StemCell Technologies). To isolate CD8⁺ T cells from CLL patient samples, CD19⁺ B cells were first depleted using EasySep B-cell isolation kit (StemCell Technologies) before undergoing CD8⁺ T cells negative selection. To further enrich memory and naïve T cells from negatively selected CD8⁺ T cells, we used biotin-conjugated antibodies against CD45RO and CD244 (2B4), which are predominantly expressed by effector and memory T cells, for positive selection of memory T cells. The flow-through fraction is untouched naïve CD8⁺ T cells. Using this approach, we can obtain high purity of naïve and memory CD8⁺ T cells (greater than 90% purity).

Wu J., Xu X., Lee E.J., Shull A.Y., Pei L., Awan F., Wang X., Choi J.H., Deng L., Xin H.B., Zhong W., Liang J., Miao Y., Wu Y., Fan L., Li J., Xu W, & Shi H. (2016). Phenotypic alteration of CD8+ T cells in chronic lymphocytic leukemia is associated with epigenetic reprogramming. Oncotarget, 7(26), 40558-40570.

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Anti-CD39 Antibody Binding Assay

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Example 9

B cells were isolated from human donor leukopak using EasySep B cell isolation kit (STEMCELL Technologies). Cyno monocytes were purified from fresh cyno blood using NHP CD14 positive selection kit (Miltenyi) and flow through was collected and stained with CD4, CD8, CD20, CD16, and CD3 antibodies (BD). Human B cells or cyno cells were incubated with serially diluted anti-CD39 antibodies (15 μg/ml 7.5 fold serial dilution, 8-point) for 30 minutes at 4 degrees C. Cell were washed 3 times in FACS buffer (PS, 2% FBS, and 2 mM EDTA) and incubated with secondary antibody (mouse anti-human IgG southern biotech) at 1:100 for 30 minutes at 4 degrees C. Cells were washed 2 times in FACS buffer and resuspended in FACS buffer and analyzed on BD Celesta.

Detection of antibody binding is as described in FIG. 7 where B cells were incubated with serially diluted antibodies and detected using a fluorescently tagged antibody and analyzed by flow cytometry. The results are shown in FIG. 7 and appear to indicate that the antibodies bind specifically to both human and cyno B cells with EC50s that range from 0.02 μg/ml to 3.18 μg/ml (human) and 0.03 μg/ml to 0.17 μg/ml (cyno). Similar binding was observed on human tumor cells lines (FIG. 3) where subset of the antibodies had a low maximal MFI and a subset had a high MFI to both the human and cyno B cells.

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Isolation and Stimulation of Splenic B Cells and DRG Neurons

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Splenic B cells were purified from 7- to 10-week-old naïve C57BL/6 mice using the EasySep B Cell Isolation Kit (STEMCELL Technologies, Cambridge, MA, USA). B cell purity (CD19-positive cells) was confirmed (>99%) using flow cytometry. Isolated B cells were cultured in complete DMEM containing penicillin, streptomycin, glutamine, β-mercaptoethanol (1× each) and 10% fetal bovine serum at 3 × 10⁶ cells/ml per well (24 well plate) in duplicate. DRG neurons were isolated from naïve C57BL/6 mice and depleted of B cells, as described above. DRG neurons from five naïve mice were cultured in 0.25 ml per well (48 well plate) in duplicate per condition in complete neurobasal media (described above) containing 10% fetal bovine serum and 1× β-mercaptoethanol. B cells and DRG neurons were stimulated with (1) vehicle (untreated cells); (2) LPS (25 µg/ml) + IL-4 (10 ng/ml); and (3) anti-CD40 (10 µg/ml) + IL-4 (10 ng/ml). B cells were harvested 24, 48, and 72 h post stimulation, and DRG neurons were harvested 48, 72, 96, and 120 h post stimulation, and markers of antibody production and the generation of plasmablasts/plasma cells were assessed by qPCR (e.g., Rag2, Aicda, and Prdm-1, as described below; see Table 1 for primers).

Gunasekaran M., Chatterjee P.K., Shih A., Imperato G.H., Addorisio M., Kumar G., Lee A., Graf J.F., Meyer D., Marino M., Puleo C., Ashe J., Cox M.A., Mak T.W., Bouton C., Sherry B., Diamond B., Andersson U., Coleman T.R., Metz C.N., Tracey K.J, & Chavan S.S. (2018). Immunization Elicits Antigen-Specific Antibody Sequestration in Dorsal Root Ganglia Sensory Neurons. Frontiers in Immunology, 9, 638.

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Splenocyte Activation and B Cell Isolation

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Splenocytes in single-cell suspensions were harvested from naive C57BL/6 wild-type mice. 1.5 × 10⁵ total splenocytes or isolated B cells were plated in 96-well flat-bottomed plates and cultured in complete RPMI with 10% FCS in vitro for 48 h in the absence or presence of 2.5 ug/ml of Resiquimod (R848) followed by analysis by flow cytometry. B cells were negatively selected using the EasySep^™ B Cell Isolation Kit (Stemcell Technologies) according to the manufacturer’s instructions. Supernatants were collected and stored at minus 80°C until assessment by ELISA.

Hägglöf T., Vanz C., Kumagai A., Dudley E., Ortega V., Siller M., Parthasarathy R., Keegan J., Koenigs A., Shute T, & Leadbetter E.A. (2022). T-bet+ B cells accumulate in adipose tissue and exacerbate metabolic disorder during obesity. Cell metabolism, 34(8), 1121-1136.e6.

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