For histology, mice were intracardially perfused with 20 ml cold PBS and fixed by perfusion with 10 ml of 4 % paraformaldehyde (PFA). Spinal cords were removed and kept in PFA for 48 h at 4 °C. The fixed spinal cords were cut into 3 mm thick transverse segments and embedded in paraffin. To evaluate demyelination, spinal cord sections were stained with Luxol Fast Blue and subsequently incubated with Periodic acid-Schiff. Immunohistochemistry was performed using the biotin-streptavidin peroxidase technique (K5001, Dako) in an immunostainer (AutostainerLink 48, Dako). Sections were pretreated in a steamer (treatment solutions pH 6.0 or pH 9.0 (Dako)) before incubation with the primary antibodies against CD3 (clone CD3-12, BioRad, 1:100) or Mac3 (clone M3/84, BD, 1:100) or B220 (clone RA3-6B2, BD, 1:200). DAB (3,3ʼ-Diaminobenzidin) was used as a chromogen. For B220/Ki67 double-immunofluorescence staining, B220 (clone RA3-6B2, BD, 1:100) and Ki67 (clone SP6, Thermo Scientific, 1:100) were used as primary antibodies; AF488- and AF594-labeled secondary antibodies (both 1:100) were used for visualization. Stained sections were analysed with a keyence microscope and pictures were taken with an Axioplot camera. ImageJ v1.48 was used to manually count infiltrated cells and measure areas.
B220 clone ra3 6b2
Manufactured by BD
Sourced in United States
The B220 is a mouse monoclonal antibody that reacts with the CD45R antigen. It is commonly used as a marker for B cells in flow cytometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 clone gk1, by BD (4 mentions)
Cd138 clone 281 2, by BD (3 mentions)
Fortessa, by BD (3 mentions)
Cd11b clone m1 70, by BD (3 mentions)
Cd69 clone h1.2f3, by BioLegend (2 mentions)
Cd23 clone b3b4, by BioLegend (2 mentions)
Cd21 35 clone 7g6, by BD (2 mentions)
Cd8α clone 53 6, by BD (2 mentions)
Cd11c clone hl3, by BD (2 mentions)
Cd19 clone 1d3, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Avidin biotin blocking kit, by Vector Laboratories (2 mentions)
5 and 6 carboxyfluorescein, by Thermo Fisher Scientific (2 mentions)
Anti mouse sn clone ser 4, by Thermo Fisher Scientific (2 mentions)
Anti mouse f4 80 clone ci a3 1, by Bio-Rad (2 mentions)
Alexa fluor 647 conjugated goat anti rat igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555 conjugated streptavidin, by Thermo Fisher Scientific (2 mentions)
Fv1000 confocal microscope, by Olympus (2 mentions)
Cd44 clone im7, by BD (1 mentions)
Cd62l clone mel 14, by BD (1 mentions)
13 protocols using b220 clone ra3 6b2
1
Histological Analysis of Spinal Cord Demyelination
2
Comprehensive Immune Cell Analysis
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Spleens and cLNs were harvested and dissociated by mechanical dispersion. Following RBC lysis with ACK Lysis Buffer (Lonza), cells were incubated with Fc block (CD16/32, clone 2.4G2, BD Biosciences) and treated with antibodies directed against the following as indicated: B220 (clone RA3-6B2, BD Biosciences), CD23 (clone B3B4, Biolegend), CD21/35 (clone 7G6, BD Biosciences), T-bet (clone 4B10, BD Biosciences), CD11c (clone HL3, BD Biosciences), CD4 (clone GK1.5, BD Biosciences), CD8α (clone 53-6.7, BD Biosciences), CD44 (clone IM7, BD Biosciences), CD62L (clone MEL-14, BD Biosciences), CD138 (clone 281-2, BD Biosciences), CD69 (clone H1.2F3, Biolegend), and TLR7 (clone A94B10, BD Biosciences). Data were acquired using a BD Biosciences Fortessa and analyzed with FlowJo software (BD Biosciences).
3
Flow Cytometric Analyses of B Cells and Tfh Cells
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For flow cytometric analyses of B cell populations, single cells were first prepared from lymph nodes by grinding with sieve. After staining with fluorescein isothiocyanate-labeled antibodies for CD19 (clone 1D3, Cat. 557398, BD Biosciences), cells were counted with BD LSRFortessa (BD Biosciences) and analyzed using BD FACSDiva software (BD Biosciences) and FlowJo software (FlowJo, Ashland, OR, USA). For flow cytometric analyses of Tfh cells, lymphocytes were stained with antibodies to CD4 (clone GK1.5, Cat. 552051, BD Biosciences), B220 (clone RA3-6B2, Cat. 562290, BD Biosciences), CD11b (clone M1/70, Cat. 563015, BD Biosciences), PD-1 (clone J43, Cat. 562584, BD Biosciences), CXCR5 (clone 2G8, Cat. 560615, BD Biosciences) and counted with BD LSRFortessa Cytometry (BD Biosciences).
4
Multiparameter Flow Cytometry Isolation
5
Comprehensive B Cell Immunophenotyping
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For flow cytometry, we used fluorochrome- or biotin-labeled antibodies against B220 (clone RA3–6B2), CD1d (clone 1B1), CD16/CD46 (Fc block; clone 2.4G2), CD19 (clone 1D3), CD21/CD35 (clone 7G6), CD23 (clone B3B4), CD35 (clone 8C12), CD38 (clone 90), CD95 (clone Jo2), CD138 (clone 281-2), GL-7 (clone GL-7), IgD (clone 11–26c.2a), IgM (clone R6–60.2), IgMa (clone DS-1), IgMb (clone AF6-78), and C3 split products (clone RmC11H9); all were purchased from BD Biosciences, Biolegend, eBioscience, or Cedarlane Laboratories. Rabbit polyclonal IgG anti-SRBC was prepared in-house (26 (link)). Staining was done in FACS buffer (PBS 2% FBS; Sigma Aldrich) or, when two or more Brilliant Violet™ antibody conjugates were used, in Brilliant Stain Buffer (BD Biosciences). All antibodies were used at ≤0.5 µg per 1x106 cells.
6
Immunophenotyping of SMG and Splenocytes
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Flow cytometry was performed as previously described (9 (link)). Briefly, SMG cells and splenocytes were dissociated as described above. Cells were incubated in Fc block [(Cd16/32, clone 2.4G2), BD Biosciences] and were treated with the following antibodies as indicated: B220 (clone RA3-6B2, BD Biosciences), TLR1 (clone CB225, BD Biosciences), TLR2 (clone 6C2, BD Biosciences), TLR4/Md2 (Clone MTS510, BD Biosciences), and TLR6 (clone C1N2, BD Biosciences). Data were acquired using a Fortessa (BD Biosciences) and analyzed using FlowJo software (BD Biosciences).
7
Visualizing GBS Infection in Mice
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All studies were approved by the UCSD Committee on the Use and Care of Animals and performed using accepted veterinary standards. Generation of Sn-deficient mice has previously been described [19 (link)]. GBS DK23 was labeled with 5-(and-6)-carboxyfluorescein (Invitrogen) per manufacturer’s instructions. Mice were intravenously infected with 2 × 108 CFU of labeled GBS via tail vein. Kidney, lung, and spleen were collected for CFU enumeration or frozen in OCT solution 1 h after infection. Spleen cryostat sections (5 μm) were fixed in acetone and blocked with 1 % BSA/PBST and AVIDIN/BIOTIN blocking kit (Vector Laboratories). The sections were stained with rat mAb anti-mouse Sn (clone SER-4) with Alexa Fluor 647-conjugated goat anti-rat IgG (Life Technologies) followed by second-step staining with biotinylated rat mAb anti-mouse F4/80 (clone CI:A3-1, Serotec) or B220 (clone RA3-6B2, BD Biosciences) with Alexa Fluor 555-conjugated streptavidin (Life Technologies). Images were from an Olympus FV1000 confocal microscope using FV1000 Viewer software.
8
Aged Murine Tumor Pathology
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Mice were aged for 600 days and terminated either when visible tumour was apparent, when they lost 20% of body weight, or showed other signs of distress, whichever was earliest. Histological examination was carried out to identify pathology, and tumours were collected and fixed in 10% neutral buffered formalin for at least 24 h followed by standard histological processing and wax embedding. Four micrometre sections were cut followed by hematoxylin and eosin (H&E) staining. Immunohistochemistry analysis was performed on tumour sections to confirm the histopathologic examination. CD3 antibody (#ab5690, 1:400, Abcam, UK) was used to detect T lymphocytes; and B220 (#clone RA3-6B2, 1:100, BD Pharmingen, USA) and paired box transcription 5 (PAX5, # 24/Pax-5, 1:100, BD Pharmingen) antibodies were used to detect B lymphocytes, respectively. All animal experiments were carried out under the respective institutional ethical approval University of Otago (AEC 03/12) and Children’s Medical Research Institute (AEC C257).
9
Visualizing GBS Infection in Mice
10
Immune Cell Depletion in Tumor Model
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Mice were administered 100 μg/dose of anti-CD8 (clone 2.43, Bio X Cell, West Lebanon, NH), anti-CD4 (clone GK1.5, Bio X Cell), anti-NK1.1 (clone PK136, Bio X Cell), or the corresponding isotype control antibody, intraperitoneally, on the day of tumor challenge. Depletion antibodies were continuously administered every 3–4 days until the end of the study. To confirm successful depletion (>95%) of the target population, blood from mice was collected, stained for B220 (clone RA3-6B2, BD Biosciences), CD3ε (clone 145–2C11, Thermo Fisher Scientific), CD8 (clone H35–17.2, Thermo Fisher Scientific), CD4 (RM4–5, Thermo Fisher Scientific), and NKp46 (29A1.4, Thermo Fisher Scientific), and analyzed by flow cytometry.
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