Single cell suspensions were generated from lymph nodes of 8–10 week old mice, resuspended at 2×107 cells/ml, rested in serum free media at 37°C for 1 hour, and stimulated in 96 well V-bottom plates at 37°C with vehicle, CpG 1668 (Invivogen) or F(ab’)2 anti-mouse IgM (Jackson Immunoresearch) for the times indicated in the figure legend. Cells were fixed with pre-warmed 1% paraformaldehyde, washed, permeabilized with ice cold 100% methanol, washed, rehydrated in PBS, stained with antibodies against pERK or IκBα (Cell Signaling Technologies), washed, stained with PE-donkey anti-rabbit IgG (Jackson Immunoresearch) and anti-B220-APC (eBiosciences), and analyzed as described above.
Anti b220 apc
Manufactured by Thermo Fisher Scientific
The Anti-B220-APC is a lab equipment product that is used for the detection and identification of B220 positive cells in flow cytometry applications. It contains an antibody conjugated with the fluorescent dye APC, which binds specifically to the B220 antigen expressed on the surface of B cells. This product provides a reliable and sensitive tool for researchers to analyze and quantify B cell populations in various biological samples.
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Lab products found in correlation
Anti igm fitc, by Thermo Fisher Scientific (4 mentions)
Anti cd43 pe, by BD (3 mentions)
Red blood cell lysing buffer, by Merck Group (3 mentions)
Anti cd4 percp, by Thermo Fisher Scientific (2 mentions)
Anti cd138 pe, by Thermo Fisher Scientific (2 mentions)
Anti gl7 fitc, by Thermo Fisher Scientific (2 mentions)
Anti b220 microbeads, by Miltenyi Biotec (2 mentions)
Anti cd4 pe, by Thermo Fisher Scientific (2 mentions)
Pna biotin, by Vector Laboratories (2 mentions)
Anti igd fitc, by BD (2 mentions)
Anti cd3 pe, by Thermo Fisher Scientific (2 mentions)
Anti iga alexa488, by Thermo Fisher Scientific (2 mentions)
Anti mouse igm f ab 2, by Jackson ImmunoResearch (1 mentions)
Pe donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Cpg 1668, by InvivoGen (1 mentions)
Facsaria 3 cell sorter, by BD (1 mentions)
Anti cd3 fitc, by Thermo Fisher Scientific (1 mentions)
Lps e coli o111 b4, by Merck Group (1 mentions)
Anti cd8 pe cy7, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
11 protocols using anti b220 apc
1
Stimulation of Lymph Node B Cells
2
Isolation of Murine Pro-B Cells
3
Flow Cytometric Analysis of B Cell Populations
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Flow cytometric analysis of B cell populations was performed using our previously described gating strategies8 (link)15 (link)16 (link)17 (link). Following red cell lysis, cells were counted and stained with a cocktail of antibodies for the detection of the indicated populations using anti-CD19-AF700, anti-CD23-PE/Cy7, anti-CD21-E450, anti-CD43-PE/Cy7, anti-GL7-FITC, anti-CD138-PE, anti-B220-E450, anti-B220-APC, anti-IL-10-APC, anti-CD3-FITC, anti-CD4-PerCP and anti-CD8-PE/Cy7 antibodies from eBioscience and Biolegend. Plasma cells were phenotyped as (Dump (CD3, CD4, CD8, CD11b, GR1 & CD11c)− CD138+CD19−B220−) with the Dump+ markers identified using biotinylated antibodies and svPE. Alternatively, splenocytes were stimulated with 50 ng/ml PMA (Sigma-Aldrich, UK) plus 500 ng/ml ionomycin (Sigma-Aldrich, UK) and 10 μg/ml LPS (E. coli O111:B4, Sigma-Aldrich, UK) for 1 h before addition of 10 μg/ml Brefeldin A (Sigma-Aldrich, UK) and cells were then incubated for a further 5 h at 37 °C with 5% CO2. Cells were fixed and washed several times in permeabilization buffer before anti-IL-10 was added in permeabilization buffer and incubated at 4 °C for 30 minutes. Cells were then washed three times with permeabilization buffer and finally with FACs staining buffer before data were acquired using a BD LSR II flow cytometer and analysis undertaken by FlowJo software (TreeStar).
4
Multiparametric Flow Cytometry of Immune Cell Subsets
5
RAG on-target and off-target analysis of IgH V(H) inversion in pro-B cells
6
Western Blot Analysis of Immune Signaling
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Tissue or cell lysates were prepared in lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS) with freshly supplemented protease inhibitors cocktail (Roche). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The individual proteins were detected using the indicated antibodies. All antibodies used in this program were as follows: anti-RIG-I (Santa Cruz), anti-RIG-I (Abcam), anti-PD1 (Thermo Scientific), anti-B220 APC (eBioscience), anti-IgA (eBioscience), anti-Akt (Cell Signaling), anti-pAkt (Ser473; Cell Signaling), anti-STAT3 (Cell Signaling), anti-pSTAT3 (Cell Signaling), anti-ERK1/2 (Cell Signaling), anti-pERK1/2 (Cell Signaling), anti-p38 (Cell Signaling), anti-p-p38 (Cell Signaling), anti-p50 (Neomarker), anti-p65 (Neomarker), anti-RelB (Santa Cruz), anti-p27 (Santa Cruz), anti-Gαi2 (Santa Cruz) and anti-PCNA (Santa Cruz).
7
Characterizing Immune Cell Subsets
8
Characterizing Immune Cell Populations in Mouse Spleen
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Spleen tissues were obtained from mice at 15–20 weeks after primary immunization, and the B- and T-cell populations of interest were identified using the following specific antibodies (eBioscience): anti-B220-APC, anti-GL7-FITC (or PerCP), anti-CXCR5-PerCP, anti-CD138-PE, anti-ICOS-PE (or APC), anti-IgD-FITC, anti-IgM-PE, anti-CD19-PE (or PerCP), anti-CD5-FITC, anti-IL-10-FITC, anti-Foxp3-PE, anti-CD4-PerCP (or FITC, PE), anti-CD25-APC, anti-Foxp3-FITC (or APC), anti-IL-17-PE, anti-IL-4-PE (or FITC), and anti-IFN-γ-FITC (or PE). Stained sections were visualized by confocal microscopy (LSM 510 Meta, Carl Zeiss, Oberkochen, Germany). The expression of GC or plasma B cells were estimated by comparing the mean fluorescence intensity using LSM 510 Meta, Carl Zeiss software.
9
RAG on-target and off-target analysis of IgH V(H) inversion in pro-B cells
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For RAG on-target and off-target analysis, single cell suspensions were derived from bone marrows of 4–6-week-old C57BL/6 WT and IgH VH inversion mice and incubated in Red Blood Cell Lysing Buffer (Sigma-Aldrich, #R7757) to deplete the erythrocytes. B220+CD43hi+IgM- pro-B cells were isolated by staining with anti-B220-APC (eBioscience, #17–0452-83), anti-CD43-PE (BD PharMingen, #553271), and anti-IgM-FITC (eBioscience, #11–5790-81) antibodies and purified by fluorescence-activated cell sorting (FACS), the purified primary pro-B cells were subjected to HTGTS-V(D)J-seq as described13 (link),14 (link).
For 3C-HTGTS, ChIP-seq and GRO-seq, B220-positive WT and IgH VH inversion primary pro-B cells were separately purified via anti-B220 MicroBeads (Miltenyi, #130–049-501) from individual 4–6-week-old RAG1-deficient mice (WT and IgH VH locus inversion) and were cultured in opti-MEM medium containing 10% (v/v) FBS plus IL-7 and SCF for 4–5 days as previously described35 (link). Purified primary pro-B cells from different mice were separately cultured in different Petri dishes. Each sample was double-checked and confirmed by PCR prior to various assays as described below. PCR primers are listed inSupplementary Table 6 .
For 3C-HTGTS, ChIP-seq and GRO-seq, B220-positive WT and IgH VH inversion primary pro-B cells were separately purified via anti-B220 MicroBeads (Miltenyi, #130–049-501) from individual 4–6-week-old RAG1-deficient mice (WT and IgH VH locus inversion) and were cultured in opti-MEM medium containing 10% (v/v) FBS plus IL-7 and SCF for 4–5 days as previously described35 (link). Purified primary pro-B cells from different mice were separately cultured in different Petri dishes. Each sample was double-checked and confirmed by PCR prior to various assays as described below. PCR primers are listed in
10
Immunofluorescence Analysis of Immune Cells
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