Splenic B cells from 8- to 12-week-old mice were purified by depletion of CD43+ cells using anti-CD43-coupled magnetic beads (Miltenyi Biotec), stained with CellTrace Violet following the CellTrace Violet Cell Proliferation Kit (Invitrogen) protocol, seeded in 96-well plates (1 × 105 per well) in 200 μl RPMI supplemented with 10% FBS, 0.05 mM 2-mercaptoethanol, 25 ng/ml recombinant mouse IL-4 (R&D Systems), and 40 μg/ml LPS (Sigma-Aldrich). On day 3 or day 5 of stimulation, B cell Fc receptors were blocked with PBS containing 2% rat serum and 10 mM EGTA and stained with FITC-conjugated anti-IgG1 (BD Biosciences) PE-conjugated IgE (BioLegend). Flow cytometry was performed using an LSRII (BD), excluding dead cells by 7-AAD staining. Data were analyzed with FlowJo software.
Anti cd43 coupled magnetic beads
Manufactured by Miltenyi Biotec
Anti-CD43-coupled magnetic beads are a laboratory product designed for cell separation and isolation applications. The beads are coated with antibodies targeting the CD43 surface antigen, allowing for the specific capture and isolation of cells expressing this marker. The magnetic properties of the beads enable their separation and manipulation using a magnetic field.
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Lab products found in correlation
Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Recombinant mouse il 4, by R&D Systems (1 mentions)
Fitc conjugated anti igg1, by BD (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Disposable micropestles, by Corning (1 mentions)
Ack buffer, by Thermo Fisher Scientific (1 mentions)
Addavax vac adx 10, by InvivoGen (1 mentions)
Imject alum, by Thermo Fisher Scientific (1 mentions)
3 protocols using anti cd43 coupled magnetic beads
1
Mouse Splenic B Cell Proliferation
2
Isolating and Analyzing Immune Cell Populations
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For flow cytometry and cell sorting, cell suspensions of LNs were obtained by mechanical disassociated with disposable micropestles (Axygen). Spleens were homogenized by filtering through a 70-μm cell strainer and red-blood cells were lysed with ACK buffer (Thermo Scientific). Samples were enriched for B cells prior to flow cytometry and sorting by negative selection using anti-CD43-coupled magnetic beads (Miltenyi Biotec). BM cells were extracted by centrifugation of punctured tibiae and femurs at up to 10,000 x G for 10 s, then treated with ACK red blood cell lysing buffer. Cells from each tissue were resuspended in PBS supplemented with 0.5% BSA and 1mM EDTA and incubated for 30 min on ice with various fluorescently-labeled antibodies (see STAR Methods Key Resources Table ). Cells were filtered and washed with the same buffer before analysis or sorting on BD FACS LSR II, FACS ARIA II, or FACS Symphony cytometers. Data were analyzed using FlowJo.
3
Adoptive Transfer of CGG-specific B Cells
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Adoptive transfer of CGG-specific B cells was performed by transferring either blood (Fig. 2 c ) or purified B cells (Fig. 2 b and Fig. 6, a and b ) from donor to recipient. For B cells transferred in blood, 100 µl of blood (corresponding to ∼4 × 105 B cells) was collected from a donor mouse and directly injected intravenously into the recipient. For transfer of purified B cells, we isolated B cells from total splenocyte preparations by negative selection using anti–CD43-coupled magnetic beads (130–090-862; Miltenyi Biotec). Untouched B cells were purified according to the manufacturer’s protocol, and 5 × 105 B cells were transferred into each recipient. 24 h following cell transfers, mice were immunized in each footpad with 10 µg CGG precipitated in 1/3 vol Imject Alum (77161; ThermoFisher Scientific; Fig. 5 ) or in each footpad and also intraperitoneally with CGG in 1/2 vol Addavax (vac-adx-10; InvivoGen; 10 µg/footpad and 20 µg intraperitoneally per mouse; Fig. 6, a and b ). To distinguish our transferred B cells, we used CD45.1 congenic mice as recipients (strain 002014; Jackson Laboratories). All protocols were approved by the Institutional Animal Care and Use Committee of the Rockefeller University.
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