Spleens and bone marrow (BM) from naïve mice were harvested from age- and sex-matched cohorts, and red blood cells were lysed by ammonium chloride lysis. Splenocytes were incubated for 30 minutes on ice with the following antibodies and dyes: viability (Zombie Aqua, Biolegend), anti-B220 (RA3–6B2, BD Biosciences), anti-CD19 (6D5, Biolegend), anti-CD23 (B3B4, Biolegend), anti-CD93 (AA4.1, Biolegend), anti-Ly77 (GL7, Biolegend), anti-IgD (11-26C.2a, Biolegend), anti-IgM (RMM1, Biolegend). and anti-F4/80 (BM8, Biolegend) . BM was incubated for 30 minutes on ice with the following antibodies and dyes: anti-B220 (RA3–6B2, BD Biosciences), anti-CD138 (281-2, Biolegend), anti-VpreB (R3, Biolegend), and anti-CD24 (M1/69, Biolegend). FCRL1 expression in different cell types in the bone marrow and spleen was tracked with anti-CD307a (REA566, Miltenyi).
Anti b220 ra3 6b2
Manufactured by BD
Sourced in United States
Anti-B220 (RA3-6B2) is a mouse monoclonal antibody that recognizes the B220 antigen, also known as CD45R. The B220 antigen is expressed on the surface of B-lymphocytes and is commonly used as a marker for the identification and analysis of B-cells in flow cytometry and other immunological applications.
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Lab products found in correlation
Anti cd4 rm4 5, by BD (5 mentions)
Anti cd11b m1 70, by BD (3 mentions)
Lasaf 2, by Leica (2 mentions)
Tcs sp5 confocal microscope, by Leica (2 mentions)
Anti cd43 s7, by BD (2 mentions)
Anti igd 11 26c 2a, by BD (2 mentions)
Anti cd11c hl3, by BD (2 mentions)
Anti cd4 gk1, by BioLegend (2 mentions)
Anti cd8 53 6, by BD (2 mentions)
Facscanto 2 cytometer, by BD (2 mentions)
Anti cd45.2 104, by BD (2 mentions)
Anti cd45 30 f11, by BD (2 mentions)
Anti igm rmm1, by BioLegend (2 mentions)
Anti cd25 pc61, by Thermo Fisher Scientific (2 mentions)
Anti cd44 im7, by BD (2 mentions)
Fluorescent mounting medium, by Agilent Technologies (1 mentions)
Streptavidin alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Lsr fortessa cytometer, by BD (1 mentions)
Anti cd23 b3b4, by Thermo Fisher Scientific (1 mentions)
Anti gl7 gl 7, by Thermo Fisher Scientific (1 mentions)
21 protocols using anti b220 ra3 6b2
1
Multiparameter Immune Cell Profiling
2
Immunofluorescence Imaging of Lymph Nodes
3
Quantification of Germinal Centers in Lymph Nodes
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Inguinal LNs (n = 3) were collected in Histocon (HistoLab) 10 days after immunization, embedded in Tissue-Tek OCT (Sakura), snap-frozen in isopentane cooled in liquid N2 and stored at −80 °C until they were cut into 7 μm sections. The sections were fixed in acetone and stored at −20 °C until staining. Three sections of different depths from each mouse were stained. Slides were blocked with 10% goat serum and stained overnight with biotinylated anti-B220 (RA3-6B2, BD Pharmingen), anti-CD4-FITC (L3T4, eBioscience) and anti-GL-7-eFluor660 (GL-7, eBioscience) at 4 °C, followed by a 1-h incubation with streptavidin-Alexa Fluor 594 (Life Technologies) at room temperature. Slides were mounted with Fluorescent Mounting Medium (Dako) and examined with a Zeiss Axio Imager.Z2 microscope. Images were acquired with Zeiss Zen Pro software. The number of GCs per section was counted under the microscope, and the GC area in each section was quantified using ImageJ 1.49 v software (NIH, USA).
4
Flow Cytometry Analysis of Bone Marrow and Spleen Leukocytes
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Single-cell leukocyte suspensions from the bone marrow and spleen were used in the flow cytometry experiments. CD16/CD32 (93, BioLegend) was used to block FcRs. The following antibodies [conjugated to eFluor 450, fluorescein isothiocyanate, phycoerythrin (PE), PE-Cy5, PerCP/Cy5.5, and PE-Cy7] were used: anti-CD21 (7G6, BD Biosciences), anti-CD23 (B3B4, eBioscience), anti-CD43 (S7, BD Biosciences), anti-CD93 (AA4.1, eBioscience), anti-CD95 (15A7, eBioscience), anti-B220 (RA3-6B2, BD Biosciences), anti-GL7 (GL-7, eBioscience), anti-IgD (11-26c.2a, BD Biosciences), and anti-IgM (II/41, eBioscience). Detection of cell surface marker expression was performed using an LSRFortessa cytometer (BD Biosciences). Typically, living lymphocytes, judged by forward and side scatter parameters, were gated for analysis. Data were further analyzed using FlowJo (Tree Star).
5
Adoptive Transfer of SWHel B Cells and TCR Transgenic T Cells
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For adoptive transfer, splenic SWHEL B cells and TCR transgenic T cells from pooled lymph nodes were co-transferred at a 5:1 ratio (1×106 B cells, 0.2×106 T cells). For flow cytometric analysis of blood, lymph node, spleen and tumor, samples were prepared as described previously [11 (link)]. The following monoclonal Abs were used to stain cells: anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD11b (M1/70), anti-NK1.1 (PK136), anti-CD45 (30-F11), anti-CD95 (Jo2), anti-Ly-77 (GL7), anti-MHCII (M5/114.15.2), anti-Ter119 (TER 119) and anti-B220 (RA3-6B2) obtained from BD Biosciences (Franklin Lakes, NJ, USA); anti-CD1d (1B1), anti-CD5 (53-7.3), anti-CD19 (6D5), anti-CD45.2 (104), anti-CD45.1 (A20), anti-Gr1 (RB6-8C5) and anti-Granzyme B (GB11) obtained from BioLegend (San Diego, CA, USA). All antibodies were directly conjugated with FITC, PE, allophycocyanin, Pacific Blue or cyanin conjugates PE-Cy7, PerCP-Cy5.5 or allophycocyanin-Cy7. Non-specific binding to Fc receptors blocked using anti-CD16/32 purified in house from the 2.4G2-hybdridoma. Intracellular staining of FoxP3 was performed using a murine FoxP3 staining kit and anti-Foxp3 mAb (FJK-16s) from eBioscience. SWHEL B cells were detected with HEL protein conjugated to Alexa647, kindly provided by Chris Jolly. Samples were analyzed on LSR-II, Fortessa and FACSCanto BD flow cytometers.
6
Murine Myeloid Progenitor Enrichment
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Bone marrow was collected by aspiration from mouse femurs and tibias and processed to generate single-cell suspensions. After red blood cell lysis, progenitors were enriched by negative selection by staining with biotinylated anti-CD4 (RM4-5; BD), anti-CD5 (53-7.3; BD), anti-CD8α (53-6.7; BD), anti-CD19 (1D3; BD), and anti-B220 (RA3-6B2; BD) followed by magnetic depletion with antibiotin microbeads (#130-090-485; Miltenyi Biotec). Enriched progenitors were plated at 5 × 105 cells/well in 24-well plates and cultured in RPMI supplemented with 10% FCS, penicillin/streptomycin, 10 mM Hepes, 2-mercaptoethanol, 10 ng/ml recombinant murine GM-CSF (Peprotech), and 5 ng/ml rmIL-4 (Peprotech). Every other day, half the medium was removed and replaced with fresh differentiation medium. After 6 d of culture, repeated pipetting was used to collect loosely adherent cells for analysis.
7
Multiparameter Flow Cytometry Analysis
8
Multiparameter Flow Cytometry Analysis of Germinal Center B Cells
9
Skin Biopsy and Cell Staining
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Biopsies of 0.6 cm in diameter were taken of the injection site, and skin was mechanically disrupted using a sterile scalpel in PBS containing 2 mM EDTA. The resulting cell suspension was stained in 0.5% FBS/PBS 2 mM EDTA using anti-B220 (RA3-6B2, BD Pharmingen), anti-Cd11c (N418, eBiosciences), anti-Cd45 (30-F11, BD Pharmingen), anti-Pdca1 (927, BioLegend), anti-Cd31 (390, eBiosciences), anti-Ki67 (SolA15, eBiosciences), and anti-BrdU (Bu20A, eBiosciences).
10
Comprehensive Immune Cell Profiling
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Peripheral blood was extracted by retroorbital bleeding and blood was collected in EDTA-coated tubes. Red blood cells were lysed with ACK buffer (Quality Biological 118-156-101) for 5 min on ice. Bone marrow was isolated the tibia, femur, and pelvic bones by spinning cleaned bones for 2 min at 8,000 rpm. Single cell suspensions of spleen and thymus were prepared in FACS buffer (3% FBS in PBS) by crushing the organ through a 70-µm strainer with a plunger. Cells were blocked with rat IgG (I8015; Sigma-Aldrich) for 20 min on ice. Staining for cell surface antigens was performed by adding fluorochrome- or biotin-conjugated antibodies on ice between 20 min and overnight on ice in the dark. The following antibodies were used in this study: anti-CD11b (M1/70), anti–Gr-1 (RB6-8C5), anti-B220 (RA3-6B2), and anti–Sca-1 (D7) all from BD; anti-CD71 (RI7217), anti–Ter-119 (116228), anti–c-Kit (CD117, 2B8), anti-CD150 (TC15-12F12.2), and anti-CD48 (HM481), all from BioLegend; and anti-CD3 (145-2C11) from Thermo Fisher Scientific.
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