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Easysep mouse b cell enrichment kit

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The EasySep Mouse B Cell Enrichment Kit is a laboratory product designed for the isolation and enrichment of mouse B cells from various cell samples. The kit utilizes a magnetic bead-based separation technology to selectively remove unwanted cells, leaving the desired B cells.

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Lab products found in correlation

Lipopolysaccharides (lps), by Merck Group (4 mentions) Anti mouse cd40, by BD (2 mentions) Pierce elisa mouse mab isotyping kit, by Thermo Fisher Scientific (2 mentions) Cell proliferation kit 1, by Roche (2 mentions) Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions) Bosc23, by American Type Culture Collection (1 mentions) Alexa 647 fluorophore, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle s medium with low glucose, by Thermo Fisher Scientific (1 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions) Normal mouse serum, by Thermo Fisher Scientific (1 mentions) Permwash, by BioLegend (1 mentions) Igg1 apc, by BioLegend (1 mentions) Cellfix, by BD (1 mentions) Perm wash, by BD (1 mentions) Alamar blue, by Merck Group (1 mentions) Anti mouse igm and igg, by Qiagen (1 mentions) Mouse cd11b positive selection kit, by STEMCELL (1 mentions) Mouse cd11c positive selection kit 2, by STEMCELL (1 mentions)

Close spelling variants of this product

EasySep kits (21 citations) EasySep™ B-cell Enrichment Kit (5 citations) EasySep negative selection mouse B cell enrichment kit (2 citations)

18 protocols using easysep mouse b cell enrichment kit

1

Adoptive Transfer of B Cell Subsets

Cited 1 time
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Transfer of AM14 (16 (link)) and B1-8 B cells (44 (link)) has been described previously. Briefly, B cells were isolated from single cell suspensions of splenocytes using the EasySep Mouse B Cell Enrichment kit (StemCell Technologies, Vancouver, Canada) per manufacturer's instructions. Purity was verified to be 90-95% by flow cytometry. 3 million AM14 B cells or B1-8 B cells containing 200,000 NP-specific cells in sterile PBS were injected i.v. into recipient mice.
Sweet R.A., Nickerson K.M., Cullen J.L., Wang Y, & Shlomchik M.J. (2017). B Cell Extrinsic Myd88 and Fcer1g Negatively Regulate Autoreactive and Normal B cell Immune Responses. Journal of immunology (Baltimore, Md. : 1950), 199(3), 885-893.
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2

B Cell Enrichment and Adoptive Transfer

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Splenocyte suspensions were made in 2% fetal calf serum (FCS), 1mM EDTA, phosphate buffered saline (PBS). B cells were purified using an EasySep Mouse B cell Enrichment Kit (Stemcell Technologies). 3-12 × 106 cells suspended in PBS per mouse (as indicated) were injected intravenously (IV) on day 0.
Ols M.L., Cullen J.L., Turqueti-Neves A., Giles J, & Shlomchik M.J. (2016). Dendritic Cells Regulate Extrafollicular Autoreactive B cells via T cells Expressing Fas and Fas Ligand. Immunity, 45(5), 1052-1065.
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3

Naive B Cell Activation and Proliferation

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Naïve B cells from splenic cell suspensions were negatively selected using EasySep Mouse B cell Enrichment Kit, following the manufacturer’s instructions (StemCell). Purified B cells were suspended in RPMI containing 10% FBS, L-glutamine, and 50μM β-mercaptoethanol (complete medium). For immunoglobulin (Ig) synthesis, naïve B cells (106 cell per ml) were cultures in complete medium alone, with LPS (20μg per ml; Sigma-Aldrich) or anti-mouse CD40 (100ng per ml; Pharmingen). Supernatants were collected after 6 days and analyzed for various Ig production by Pierce ELISA Mouse mAb Isotyping Kit, following the manufacturer’s instructions (Thermo Scientific). Proliferation was measured using Cell Proliferation Kit I (MTT), following the manufacturer’s instructions (Roche). For proliferation, aliquots of 105 B cell in 100μl of complete medium alone, or in presence LPS (20μg per ml) or anti-mouse CD40 (100ng per ml) were cultured in a 96-well flat-bottom plate for 48 hours, then the MTT labeling reagent was added to a final concentration 0.5mg per ml following by overnight incubation with the solubilization solution. Proliferation was assessed by measuring the absorbance using a microplate (ELISA) reader.
Li J., Jørgensen S.F., Maggadottir S.M., Bakay M., Warnatz K., Glessner J., Pandey R., Salzer U., Schmidt R.E., Perez E., Resnick E., Goldacker S., Buchta M., Witte T., Padyukov L., Videm V., Folseraas T., Atschekzei F., Elder J.T., Nair R.P., Winkelmann J., Gieger C., Nöthen M.M., Büning C., Brand S., Sullivan K.E., Orange J.S., Fevang B., Schreiber S., Lieb W., Aukrust P., Chapel H., Cunningham-Rundles C., Franke A., Karlsen T.H., Grimbacher B., Hakonarson H., Hammarström L, & Ellinghaus E. (2015). Association of CLEC16A with human common variable immunodeficiency disorder and role in murine B cells. Nature communications, 6, 6804.
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4

Retroviral Transduction of Primary B Cells

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Retroviruses were produced by transfecting retroviral packaging cell line BOSC23 (ATCC) with individual MSCV vectors as previously described [41 (link)]. Primary B cells were isolated from naïve C57BL6/J mouse spleens (8–12 weeks of age) by negative selection using the EasySep Mouse B cell Enrichment Kit (Stem Cell Technologies) per manufacturer’s instructions. Following overnight stimulation with LPS at 20-25ug/mL, primary B cells were transduced with retroviruses supplemented with 5ug/mL polybrene by spinoculation at 2500rpm for one hour at 30°C. Cells were analyzed by flow cytometry using three wells per condition at days 2–5 post-transduction. Supernatants were collected and stored at -80°C for subsequent analysis by ELISA. IL10 in primary B cell supernatants was measured using the BD OptEIA Mouse IL-10 ELISA Kit (BD biosciences) per manufacturer’s instructions.
Terrell S, & Speck S.H. (2017). Murine gammaherpesvirus M2 antigen modulates splenic B cell activation and terminal differentiation in vivo. PLoS Pathogens, 13(8), e1006543.
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5

Isolation and Purification of B-2 and B-1a Cells

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To obtain B-2 cells, spleens were mechanically dissociated on 40-µm nylon cell strainers, followed by negative selection with the EasySep Mouse B Cell Enrichment Kit, which depleted CD43+ cells (Stemcell Technologies). B-2 cell purity was >90% as determined by flow cytometry. To obtain B-1a cells, peritoneal cells were harvested by injecting 7 mL RPMI, no phenol (Lonza), plus 3% newborn calf serum into the PerC. CD19+ CD22+ CD5+ B-1a cells were sorted as previously described to ∼95% purity (23 (link)). For B-2 versus B-1a experiments, 5 × 106 B-2 and B-1a cells were injected intraperitoneally. For other transfer experiments, 3 × 106 cells were used.
Shen L., Chng M., Alonso M.N., Yuan R., Winer D.A, & Engleman E.G. (2014). B-1a Lymphocytes Attenuate Insulin Resistance. Diabetes, 64(2), 593-603.
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6

Murine Model of TCL1-Driven Leukemia

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Eight-week-old male syngeneic immunocompetent C57BL/6 mice were injected i.p. (day 0) with 10×106 cells purified from the spleen of leukemic male Eμ-TCL1 transgenic mice using the EasySep mouse B-cell enrichment kit (STEMCELL Technologies). The purity of the transplanted CD19+ CD5+ Igκ+ cells was assessed by flow cytometry. Mice were monitored weekly for weight and leukemia development by flow cytometric analysis of PB samples. Mice were i.v. injected with 0.15mg/kg trabectedin every week starting when the frequency of CD19+CD5+ leukemic cells in the PB was 15–20%, compared to C57BL/6 wild-type mice. Mice were monitored weekly for weight and leukemia development by flow cytometric analysis of the PB samples and, depending on the last trabectedin injection, humanely killed at different time points. PE, PB, and organs (SP, LN, femoral BM) were collected and analyzed.
Banerjee P., Zhang R., Ivan C., Galletti G., Clise-Dwyer K., Barbaglio F., Scarfò L., Aracil M., Klein C., Wierda W., Plunkett W., Caligaris-Cappio F., Gandhi V., Keating M.J, & Bertilaccio M.T. (2019). Trabectedin reveals a strategy of immunomodulation in chronic lymphocytic leukemia. Cancer immunology research, 7(12), 2036-2051.
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7

Isolation and Characterization of Idiotype-Negative B Cells

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Fluorescently labeled B220+ idiotype− B cells were sorted from the spleen of a Rag−/−564Igi mouse by flow cytometry using the MoFlo sorter. Cells were stained for flow cytometry according to standard procedures. B cells were stained with B6-256 anti-Id, as described [24 (link)], which was coupled to the Alexa-647® fluorophore according to the manufacturer’s instructions (Invitrogen Molecular Probes). B cells were also stained with an anti-CD45 (B220) antibody labeled with R-Phycoerythrin (PE) from Southern Biotech as a marker for B cells. Fluorescent antibodies were generally used at 1 µg/ml. B220+ GFP− from 564Igi, 564Igi mb1-cre, 564Igi CD19cre and 564Igi CD21-cre BM and spleen cells were sorted and RNA prepared as described below. B cells from the spleen of an Aicda−/−564Igi mouse were purified using the EasySep Mouse B-Cell Enrichment kit (StemCell 19754), which isolates B cells by magnetic negative selection.
Umiker B.R., McDonald G., Larbi A., Medina C.O., Reth M, & Imanishi-Kari T. (2014). In a SLE mouse model the production of IgG autoantibody requires expression of activation-induced deaminase in early developing B cells. European journal of immunology, 44(10), 3093-3108.
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8

Isolation and Culture of Mouse Stem Cells and B Cells

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MSCs were isolated from C3H HeN (Orient, Seongnam, Korea) bone marrow according to the subfractionation culturing method.39 (link) MDFs were isolated from C3H HeN dermal tissue. These two cell lines were incubated using Dulbecco's modified Eagle's medium with low glucose (Gibco, Carlsbad, CA, USA) and supplemented with 10% fetal bovine serum (Gibco) and 1 × antibiotic-antimycotic solution (Gibco) at 37 °C in 5% CO2. B cells were isolated from Balb/c splenocytes using the EasySep Mouse B Cell Enrichment Kit (StemCell Technologies, Vancouver, BC, Canada) and cultured in RPMI 1640 (Hyclone, South Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco), 2-mercaptoethanol (Gibco), and 1 × antibiotic-antimycotic solution (Gibco).
Lee D.S., Yi T.G., Lee H.J., Kim S.N., Park S., Jeon M.S, & Song S.U. (2014). Mesenchymal stem cells infected with Mycoplasma arginini secrete complement C3 to regulate immunoglobulin production in b lymphocytes. Cell Death & Disease, 5(4), e1192-.
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9

Naive B Cell Activation and Proliferation

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Naïve B cells from splenic cell suspensions were negatively selected using EasySep Mouse B cell Enrichment Kit, following the manufacturer’s instructions (StemCell). Purified B cells were suspended in RPMI containing 10% FBS, L-glutamine, and 50μM β-mercaptoethanol (complete medium). For immunoglobulin (Ig) synthesis, naïve B cells (106 cell per ml) were cultures in complete medium alone, with LPS (20μg per ml; Sigma-Aldrich) or anti-mouse CD40 (100ng per ml; Pharmingen). Supernatants were collected after 6 days and analyzed for various Ig production by Pierce ELISA Mouse mAb Isotyping Kit, following the manufacturer’s instructions (Thermo Scientific). Proliferation was measured using Cell Proliferation Kit I (MTT), following the manufacturer’s instructions (Roche). For proliferation, aliquots of 105 B cell in 100μl of complete medium alone, or in presence LPS (20μg per ml) or anti-mouse CD40 (100ng per ml) were cultured in a 96-well flat-bottom plate for 48 hours, then the MTT labeling reagent was added to a final concentration 0.5mg per ml following by overnight incubation with the solubilization solution. Proliferation was assessed by measuring the absorbance using a microplate (ELISA) reader.
Li J., Jørgensen S.F., Maggadottir S.M., Bakay M., Warnatz K., Glessner J., Pandey R., Salzer U., Schmidt R.E., Perez E., Resnick E., Goldacker S., Buchta M., Witte T., Padyukov L., Videm V., Folseraas T., Atschekzei F., Elder J.T., Nair R.P., Winkelmann J., Gieger C., Nöthen M.M., Büning C., Brand S., Sullivan K.E., Orange J.S., Fevang B., Schreiber S., Lieb W., Aukrust P., Chapel H., Cunningham-Rundles C., Franke A., Karlsen T.H., Grimbacher B., Hakonarson H., Hammarström L, & Ellinghaus E. (2015). Association of CLEC16A with human common variable immunodeficiency disorder and role in murine B cells. Nature communications, 6, 6804.
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10

Murine Primary B Cell Isolation and LPS Stimulation

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Murine primary B cells were isolated by immunomagnetic negative selection using the EasySep Mouse B Cell Enrichment Kit (Stem Cell Technologies) as per manufacturer’s instructions. The purity of B cell isolation was routinely analyzed and found to be ≥95% as determined by staining for CD19 by flow cytometry. The cells were plated at 1×106 cells/mL of a 24 well plate overnight with cRPMI containing 20–25 µg/mL LPS (Sigma) prior to retroviral transduction.
Rangaswamy U.S., O’Flaherty B.M, & Speck S.H. (2014). Tyrosine 129 of the Murine Gammaherpesvirus M2 Protein Is Critical for M2 Function In Vivo. PLoS ONE, 9(8), e105197.
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