Single cell suspensions of spleen and mesenteric and peripheral lymph nodes from young 6-12 weeks old adult or >90 weeks old aged B1-8i mice were obtained by pressing the tissues through a 70 μm mesh in PBS with 2% foetal bovine serum under sterile conditions. B cells were then enriched using the MagniSort Mouse B cell Enrichment Kit (#8804-6827-74 Thermo Fisher Scientific), according to the manufacturer's instruction. Cell numbers and viability were determined using a CASY TT Cell Counter (Roche). A small aliquot of enriched B cells was taken and stained to determine the percentage of NP-binding B cells by flow cytometry before cell transfer. The cell suspensions were then diluted in appropriate volumes of PBS to obtain a final concentration of 1 x 105 NP-binding B cells/mL. 100μL of 1 x 104 NP-binding B cells from young and aged donor B1-8i-Tg mice were injected intravenously into the tail of congenic WT recipients. Recipient mice were then immunised subcutaneously with NP-KLH/Alum, as detailed below, and draining inguinal LNs (iLNs) were collected at the indicated time points for flow cytometry.
Magnisort mouse b cell enrichment kit
Manufactured by Thermo Fisher Scientific
The MagniSort Mouse B cell Enrichment Kit is a laboratory tool used to isolate and enrich mouse B cells from a mixed cell population. It utilizes magnetic beads coated with antibodies to selectively bind and separate B cells from other cell types, enabling their further analysis or downstream applications.
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Lab products found in correlation
Casy tt cell counter, by Roche (2 mentions)
Anti b220 antibody, by BioLegend (2 mentions)
Anti cd40 hm40 3 agonistic antibody, by Thermo Fisher Scientific (2 mentions)
Mil 4, by R&D Systems (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Facscalibur, by BD (2 mentions)
Rpmi culture medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin amphotericin b solution, by Sartorius (1 mentions)
Lps from escherichia coli, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Rpmi 1640, by Welgene (1 mentions)
Cellquest, by BD (1 mentions)
Cpg1826, by InvivoGen (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
Retinoic acid, by Merck Group (1 mentions)
Cellquest software, by BD (1 mentions)
8 protocols using magnisort mouse b cell enrichment kit
1
Adoptive Transfer of Aged B Cells
2
Isolation of B-cells and Tumor Cells from Mouse Models
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B-cells from 8-week-old male wild-type or Tg(IghMyc)22Bri (“Eµ-Myc”) mice with C57BL/6JRj background119 (link) were isolated using negative selection technique (MagniSort Mouse B cell Enrichment Kit, Thermo Fisher Scientific). IgM-positive B-cells were isolated from spleens using a standard antibody cocktail provided in the kit, and IgM-negative B-cells were isolated from bone marrow using the standard antibody cocktail with addition of biotinylated anti-IgM antibody (clone R6-60.2; 1:100; # 553406; BD Biosciences). Tumor cells were isolated from inguinal and axial lymph nodes of male and female tumor-bearing Eµ-Myc transgenic mice of 15 to 52 weeks of age. In brief, single cell suspensions were obtained by mechanical disruption in PBS pH 7.4, filtered and centrifuged for 5 min at 700×g. Supernatants were removed and resuspended in red blood cell lysis buffer (8.3 g/l ammonium chloride in 0.01 M Tris-HCl pH 7.4). Target cells were isolated according to the manufacturer’s protocol. Purity and IgM status of isolated cells were confirmed by flow cytometry (anti-CD19-APC; clone 1D3; 1:1000; # 152410; BioLegend and anti-IgM-PE-Cy7; clone eB121-15F9; 1:100; # 25-5890-82; Thermo Fisher Scientific).
3
Aged B Cell Transfer and Immune Response
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Single-cell suspensions of spleen and mesenteric and peripheral lymph nodes (LNs) from young 6- to 12-wk-old adult or >90-wk-old aged B1-8i mice were obtained by pressing the tissues through a 70-µm mesh in PBS with 2% FBS under sterile conditions. B cells were then enriched using the MagniSort Mouse B cell Enrichment Kit (8804-6827-74; Thermo Fisher Scientific), according to the manufacturer’s instruction. Cell numbers and viability were determined using a CASY TT Cell Counter (Roche). A small aliquot of enriched B cells was taken and stained to determine the percentage of NP-binding B cells by flow cytometry before cell transfer. The cell suspensions were then diluted in appropriate volumes of PBS to obtain a final concentration of 1 × 105 NP-binding B cells per milliliter. A total of 100 μl of 1 × 104 NP-binding B cells from young and aged donor B1-8i-Tg mice were injected i.v. into the tail of congenic WT recipients. Recipient mice were then immunized s.c. with NP–keyhole limpet hemocyanin (KLH; NP-KLH)/Alum, as detailed later, and draining inguinal LNs (iLNs) were collected at the indicated time points for flow cytometry.
4
Isolation and Culture of Murine B Cells
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B cells were isolated from the BM and spleen by negative selection using the MagniSort Mouse B cell Enrichment Kit (Cat. 14-0161-82; Thermo Fisher Scientific) according to the manufacturer’s protocol. The purity of the isolated cells was routinely above 95%, as assayed by flow cytometry. B cells were isolated from the peritoneal cavity as described elsewhere (Ray & Dittel, 2010 (link)). The resulting untouched, negatively selected B cells were grown in RPMI culture medium (Gibco), containing 10 mM HEPES (HyClone), 1 mM sodium pyruvate (HyClone), MEM non-essential amino acids (HyClone), 10% heat-inactivated FBS (HyClone), 1% penicillin-streptomycin-amphotericin B solution (Biological Industries), and were maintained at 37°C in a 5% CO2 atmosphere.
5
Plasma Cell Differentiation from Mouse B Cells
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Splenocytes were isolated from C57BL/6 mice and negatively selected by the MagniSort™ mouse B cell enrichment kit (Thermo Fisher Scientific) to acquire total mouse B cells. Separated B cells were stimulated by LPS from Escherichia coli (10 μg/ml, Sigma-Aldrich) for 3 days. B cells were cultured in RPMI 1640 (Welgene) with 10% fetal bovine serum (FBS), 1% antibiotics (Thermo Fisher Scientific), and 50 μM β-mercaptoethanol (Sigma-Aldrich). CD138+B220low plasma cells were analyzed by flow cytometry. IgM produced during plasma cell differentiation was measured by ELISA (Thermo Fisher Scientific). For in vivo plasma cell differentiation by LPS, LPS (0.6 mg/kg) was intravenously injected into 8-12 week-old C57BL/6 mice. On day 4 after LPS injection, mice were euthanized, and the plasma cell population in the splenocytes was analyzed using flow cytometry.
6
Purification and Stimulation of Murine Splenic B Cells
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Mouse splenic B cells were purified by negative selection (eBioscience Magnisort Mouse B cell enrichment kit). B-cell purity was >95% as measured by FACS analysis (FACSCalibur and CellQuest software; BD Biosciences) using anti-B220 antibody (BioLegend). Purified B cells were seeded at a final concentration of 0.5 or 0.25 × 106 cells/mL. For plasmablast differentiation, B cells were stimulated with 5 µg/mL LPS (Sigma) for 72 h, and for IgG1 CSR, B cells were stimulated 5 µg/mL anti-CD40 (HM40-3) agonistic antibody (eBioscience), or 5 µg/mL LPS (Sigma), together with 5 ng/mL mIL-4 (R&D Systems) for 96 h. All B cells were cultured in RPMI 1640 supplemented with 10% (vol/vol) heat-inactivated FCS, 5 mM Hepes, 2 mM l -glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM 2-mercaptoethanol.
7
Cytokine Profiling and B Cell Activation
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Peripheral blood samples were collected for serum preparation. The levels of IL-2 (171-G5003M), IL-4 (171-G5005M), IL-6 (171-G5007M), IL-17 (171-G5013M), IFN-γ (171-G5017M), and TNF (171-G5023M) were determined by Bio-Plex (Biorad) according to the manufacturer's conditions. B cells were purified from spleen with the MagniSort Mouse B cell Enrichment Kit (eBioscience), cultured in complete medium (RPMI 1640 medium supplemented with 10% FCS, sodium pyruvate, L-glutamine, antibiotics and β-mercaptoethanol) and stimulated overnight with 20 μM CpG1826 (Invivogen) as described (47 (link)). The level of IL-10 (171G5009M) in medium was determined by Bio-Plex (Biorad) according to manufacturer's conditions.
8
Induction of Immunoglobulin Class Switching in Murine B Cells
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