To isolate B cells, tonsils were obtained from patients that underwent tonsillectomy at the Hospital Universitari Germans Trias i Pujol (HUGTiP) to be anonymously used for research purposes. All participants and/or their legal tutor gave written informed consent to participate. The study protocol followed the principles of the Declaration of Helsinki and was approved by the Clinical Research Ethics Committee of our institution (Comitè Ètic d’Investigació Clínica, Refs CEIC: PI-16-056 from 5 May 2016). Tonsils were mechanically disrupted and filtered through a 70 μm cell strainer, to then isolate mononuclear cells by density gradient separation with Ficoll-Paque (GE Healthcare, Uppsala, Sweden). Mononuclear cells were frozen in 90% heat-inactivated fetal bovine serum (Lonza) and 10% Dimethyl sulfoxide (DMSO) and stored at -190°C until further use. Upon thawing, resting B cells were isolated by negative selection by labelling anti-CD43 magnetic beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Purity was confirmed by Flow cytometry (FACS Canto II, BD Bioscience, San Jose CA), labelling cells with the following antibodies; 7AAD (BD Biosciences), CD3-PE (BD Biosciences, Clone UCHT1) and CD19-BV510 (BD Horizon, clone SJ25C1), ensuring CD19 purities above 95%.
Anti cd43 magnetic beads
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD43 magnetic beads are a laboratory product designed for the isolation and enrichment of CD43-positive cells. They consist of superparamagnetic particles coated with antibodies specific to the CD43 cell surface antigen. These beads can be used to separate and enrich target cell populations from complex samples, such as peripheral blood or bone marrow, using magnetic separation techniques.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Cd4 t cell negative isolation kit, by Miltenyi Biotec (3 mentions)
Facsmelody, by BD (3 mentions)
Facscanto 2, by BD (2 mentions)
Ficoll paque, by Cytiva (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions)
Interleukin 4 (il 4), by R&D Systems (2 mentions)
Fetal calf serum (fcs), by PAN Biotech (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Iscove s medium, by Harvard Bioscience (2 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Ecl reagent, by Sartorius (2 mentions)
Sds page, by Bio-Rad (2 mentions)
Streptavidin magnetic beads, by Miltenyi Biotec (2 mentions)
Cd3 pe, by BD (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
7 aad, by BD (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
32 protocols using anti cd43 magnetic beads
1
Isolation of Resting B Cells from Tonsils
2
Splenic B Cell Isolation Protocol
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Spleens were obtained from post-mortal kidney donors (Erasmus MC Hospital, Rotterdam) and anonymously used for research purposes as described in article 13 of The Netherlands law of organ donation (Wet op Orgaandonatie, WOD). All samples and data were analyzed anonymously. Spleens were mechanically disrupted and filtered through a 70-µm cell strainer (Greiner Bio-one, Alphen a/d Rijn, The Netherlands) to obtain a single-cell suspension. Mononuclear cells, isolated using Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient, were stored at −150°C until use. Upon thawing, quiescent B cells were isolated by negative selection using anti-CD43-magnetic beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) (23 (link)). Purity was determined by flow cytometry (FACS Canto II). Typically, cell suspensions consisted of >98% pure CD19+ B cells. B cells from spleens from 12 different donors were used it the experiments.
3
Enrichment and Activation of Murine B Cells
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Mature, resting B cells were obtained from mouse spleens by forcing tissue through a 70-µm mesh into PBS containing 2% heat-inactivated fetal bovine serum (FBS). After ammonium-chloride-potassium buffer lysis for 3 min, untouched B cells were enriched using anti-CD43 magnetic beads according to the manufacturer’s protocol (Miltenyi Biotec) obtaining >95% purity. 3.2 × 107 cells/10 cm dish (Gibco) were cultured at 37°C 5% CO2 in 10 ml mouse B cell medium consisting of RPMI-1640, supplemented with 10% heat-inactivated FBS, 10 mM Hepes, antibiotic-antimycotic (1×), 1 mM sodium pyruvate, 2 mM L-glutamine, and 53 µM 2-mercaptoethanol (all from Gibco) and activated with 2 µg/ml anti-mouse RP105 clone RP/14 (produced in house or 562191; BD Pharmingen).
NB-21 feeder cells (Kuraoka et al., 2016 (link)) were maintained in DMEM supplemented with 10% heat-inactivated FBS and antibiotic-antimycotic (1×). For co-culture, feeder cells were irradiated with 80 Gy and seeded simultaneously with B cells, 24 h after transfection, into B cell culture medium supplemented with 1 ng/ml recombinant mouse IL-4 (214–14; PeproTech) and 2 µg/ml anti-mouse RP105 clone RP/14.
NB-21 feeder cells (Kuraoka et al., 2016 (link)) were maintained in DMEM supplemented with 10% heat-inactivated FBS and antibiotic-antimycotic (1×). For co-culture, feeder cells were irradiated with 80 Gy and seeded simultaneously with B cells, 24 h after transfection, into B cell culture medium supplemented with 1 ng/ml recombinant mouse IL-4 (214–14; PeproTech) and 2 µg/ml anti-mouse RP105 clone RP/14.
4
Murine B Cell Class Switching Assay
5
Isolation and Stimulation of Splenic B Cells
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Total splenocytes were isolated from 8-wk-old to 12-wk-old C57BL/6 mice (female or male). Splenic B cells were enriched by MACS depletion of CD43+ cells using anti-CD43 magnetic beads (Miltenyi Biotech) according to the manufacturer’s instructions. The purified splenic B cells were cultured for a minimum of 2 h at 37°C in 7.5% CO2 in Iscove’s medium (Biochrom, with stable glutamine) supplemented with 10 units/mL penicillin/streptomycin, 50 mM beta-mercaptoethanol (Sigma-Aldrich), and 10% FCS (PAN Biotech). The splenic B cells were then further analyzed for IgM- and IgD-BCR surface expression or stimulated with antibodies, and the recruitment of Syk to CD79a by bPHA was detected.
6
Lymphocyte Development and CSR Analysis
7
Splenic B cell expansion assay
8
Adoptive Transfer of B1-8 B Cells
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B cells and T cells were purified by forcing spleen tissue through mesh into PBS or RPMI containing 2% serum and 1 mM EDTA. Resting B cells and T cells were purified using anti-CD43 magnetic beads or CD4+ T cell–negative isolation kits (Miltenyi), respectively. After purification, 2–6 × 106 B1-8hi or B1-8lo B cells (comprising 15% and 3%, respectively, Igλ+ NP-specific B cells) or OT-II T cells were transferred intravenously into host mice before immunization. In competition experiments, the ratio of adoptively transferred cells (ICAM-1/2+/+ and ICAM-1/2−/− B1-8hi B cells) was 50:50. For plasmablast analysis, 0.5 × 106 OT-II T cells were cotransferred with the B1-8hi B cells (Fig. 2 D ).
9
Isolation and Activation of Resting B Cells
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For B cells isolation, resting B cells were purified from spleen by negative selection with anti-CD43 magnetic beads or positive selection with anti-B220 magnetic beads (MiltenyiBiotech) following the manufacturer’s instructions. The purified B cells population was >95% B220 positive cells. B cells were cultured in RPMI supplemented with 10% FCS, 2-ME, penicillin (100 U/ml), and streptomycin (100 μg/ml). Purified B cells (2×106/ml) were cultured with LPS (10 μg/ml) or IgM-specific goat F(ab’)2 antibody (10 μg/ml) in a 48-well flat-bottom plate.
10
Isolation and Stimulation of Naive Splenic B Cells
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Naive splenic B cells were isolated using anti-CD43 magnetic beads (Miltenyi Biotec; 130-049-801). Purified B cells were kept unstimulated, or stimulated with 10 μg/m LPS for 3 days. The cells were then lysed in radioimmunoprecipitation assay buffer (10 mM Tris-HCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton-100 140 mM NaCl, 0.1% deoxycholate, 0.1% SDS). Lysates were centrifuged for 15 min at 4 °C. Cleared lysates were boiled with sample buffer for 5 min, separated by SDS-PAGE (Bio-Rad), and transferred to nitrocellulose membranes. Blots were blocked with 5% skim milk in TBST and 0.05% Tween-20 for 1 h at room temperature, and incubated with primary antibody diluted 1:1000 overnight at 4 °C. Horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody and ECL Reagent (Biological Industries) were used for detection. All antibodies are listed in Table 1 .
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