As described previously35 , subcutaneous tumours with KP1.9 cells were harvested from C57BL/6 flanks 3 weeks after implantation, minced, and shaken at 600 r.p.m. with 0.2 mg ml−1 collagenase type I (Worthington Biochemical Corporation) in RPMI-1640 for 30 min at 37 °C. Digested samples were filtered (70 μm BD Falcon strainer); washed in PBS with 0.5% BSA and 2 mM EDTA; incubated with Fc-block (TruStain fcX anti-mouse CD16/32; clone 93; Biolegend) for 15 min at 4 °C; and labelled with antibodies as indicated for 45 min at 4 °C. Flow cytometry (LSRII, BD Biosciences) labelled tumour cells (CD45− EpCAM+), TAM (CD45+ CD11b+ Ly6C- Lin- CD11c+ F4/80+), lymphocyte-like cells (CD45+ CD11b- Lin+), along with CD45- EpCAM- host-cell populations. Antibodies included EpCAM (clone G8.8; eBioscience); CD45 (clone 30-F11; Biolegend), F4/80 (clone BM8; Biolegend), CD11c (clone N418; Biolegend), Ly6C (clone HK1.4; Biolegend); and CD11b (clone M1/70; BD Biosciences). The lineage (Lin) antibody mix contained anti-CD90.2 (clone 53–2.1), anti-B220 (clone RA3-6B2), anti-NK1.1 (clone PK136), anti-CD49b (clone DX5), anti-Ter119 (cloneTER-119) and anti-Ly6G (clone 1A8) (all BD Biosciences). 7-aminoactinomycin D (7-AAD, Sigma Aldrich) excluded dead cells. VT680 fluorescence was directly assessed using the LSRII flow cytometer, FlowJo v.8.8.7 (Tree Star, Inc.) and MATLAB.
Anti b220 clone ra3 6b2
Manufactured by BD
Sourced in Australia, United States
Anti-B220 (clone RA3-6B2) is a monoclonal antibody that binds to the B220 (CD45R) antigen expressed on the surface of B-lymphocytes. It can be used for the identification and isolation of B-cells in flow cytometry and other immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Axioplan 2 microscope, by Zeiss (2 mentions)
Dakocytomation target retrieval solution, by Agilent Technologies (2 mentions)
Fc block, by BD (2 mentions)
Anti ly6g clone 1a8, by BD (1 mentions)
Anti nk1.1 clone pk136, by BD (1 mentions)
Trustain fcx anti mouse cd16 32 clone 93, by BioLegend (1 mentions)
Epcam clone g8.8, by Thermo Fisher Scientific (1 mentions)
Anti cd90 2 clone 53 2, by BD (1 mentions)
Lsrii flow cytometer, by Tree Star (1 mentions)
Cd11b clone m1 70, by BD (1 mentions)
Cd11c clone n418, by BioLegend (1 mentions)
Ly6c clone hk1, by BioLegend (1 mentions)
F4 80 clone bm8, by BioLegend (1 mentions)
7 aminoactinomycin d 7 aad, by Merck Group (1 mentions)
Cd45 clone 30 f11, by BioLegend (1 mentions)
Cd11b, by BD (1 mentions)
Collagenase type 1, by Worthington (1 mentions)
F4 80, by BD (1 mentions)
Anti cd25 clone pc61, by BioLegend (1 mentions)
Anti cd4 clone rm4 5, by BioLegend (1 mentions)
11 protocols using anti b220 clone ra3 6b2
1
Tumor Cell and Immune Cell Profiling
2
Flow Cytometry Analysis of Mouse Immune Cells
3
Multicolor Flow Cytometry Analysis
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Multicolor flow cytometry for analysis or for sorting was performed on an LSR Fortessa-5 or FACS Aria (BD Biosciences), respectively. Single-cell suspensions of splenocytes were blocked with anti-CD16/32 mAb (clone 2.4G2), followed by staining with the following antibodies: anti-B220 (clone RA3-6B2) and anti-CD45.2 (clone 104) from BD Biosciences. HEL-binding B cells were stained as described previously (Chan et al, 2009 (link)). For cell cycle analyses, spleen cells were first stained for extracellular antigens and then were analysed with 10 μg/ml DAPI staining using a Cytofix/Cytoperm kit (BD Biosciences) or PFA and Tween-20. Cell cycle was calculated by FlowJo Dean/Jett/Fox algorithm or by setting gates manually. The Click-iT EdU Alexa Fluor 488 Imaging kit and CaspGLOW Fluorescein Active Caspase Staining kit (both from Thermo Fisher Scientific) were used according to the manufacturer’s instructions. Data were analysed with FlowJo software (Tree Star).
4
Lung and Lymph Node Imaging After Vaccination
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Mice were vaccinated via the i.t. route with 1 mg Advax-Fluorescein/3 μg CysVac2 (unconjugated or conjugated to AF647) as previously described, and euthanized at 1, 3 and 22 days post-vaccination. Lungs were prepared for imaging as described previously.48 (link) mLNs were isolated from the same mice, transferred straight into 4% paraformaldehyde and processed for cryotome cutting as previously described. Sections were cut to 20 µm thickness for immunohistology and stained with anti-CD11c (clone 145-2C11, Biolegend, Australian Biosearch, NSW, Australia), anti-B220 (clone RA3-6B2, BD Biosciences, NSW, Australia), anti-CD3 (clone 145-2C11, BD Biosciences, NSW, Australia) and NucBlue Live ReadyProbes Reagent (Invitrogen, Thermo Fisher, NSW, Australia).
5
Immune Cell Phenotyping in Mice
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Single cell suspensions were prepared from minced pieces of peripheral lymph nodes (pooled axial and inguinal nodes), thymus, and spleen by mechanical teasing through a metal meshwork and bone marrow cells by flushing the femurs. Blood was collected by cardiac puncture. Erythrocytes were removed from spleen and blood by hypotonic lysis. Leukocyte phenotyping was done using mAbs against CD4 (clone RM4-5, Alexa Fluor 647 conjugated), CD8 (clone 53-6.7, PerCP-Cy5.5 conjugated), and anti-B220 (clone RA3-6B2, Pacific blue conjugated), all obtained from BD Biosciences. Regulatory T cells were identified using mouse regulatory T cell staining kit (eBioscience; CD4-FITC, CD25-allophycocyanin, and FoxP3-PE). Cells were analyzed using LSRII using BDFACS-Diva software. Representative dot blots and histograms were made with FlowJo software. Frozen sections of thymuses were also stained for CD8, CD4, and CD25.
6
Lung Tissue Immunofluorescence Analysis
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Immunofluorescence staining was done as described before (2 (link)). FFPE lung sections were cut, immersed in xylene, and then hydrated in alcohol and PBS. Antigens were unmasked using DakoCytomation target retrieval solution (Dako), and non-specific binding was blocked by adding 5% (vol/vol) normal donkey serum and Fc block (BD). Avidin was used to neutralize endogenous biotin, followed by incubation with biotin (Sigma-Aldrich). Sections were then probed with anti-B220 (clone RA3-6B2, BD) to detect B cells. For analysis of B-cell follicles, follicles were outlined with an automated tool of the Zeiss Axioplan 2 microscope (Zeiss), and total area and average size were calculated in squared microns.
7
Immunofluorescent Staining of Splenic Tissue
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Mice were IV injected with liposomes, and spleens were harvested after 2 h. Subsequently, the splenic tissue was cryopreserved in liquid nitrogen and cut into slices of 5–6 µm thickness using a CryoStar NX70 (Thermo Fischer Scientific). The tissue sections were blocked using 10% normal goat serum in PBS and stained with anti-CD169 (clone SER-4, in-house produced), anti-B220 (clone RA3-6B2, BD Biosciences, San Jose, CA, USA) and DAPI. The sections were analyzed using a fluorescent microscope (Zeiss, Oberkochen, Germany) and processed using the Fiji software v1.51.
8
Immunohistochemical Analysis of Tissue Sections
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Paraffin sections (10 μm) were dewaxed and stained histochemically with hematoxylin and eosin (HE). For immunohistochemistry, the paraffin sections were dewaxed and subjected to a heat-induced epitope retrieval step except for sections for prior incubation with anti-B220 (clone RA3-6B2, BD Bioscience, 1:400). Primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, USA, 1:400) and MPO7 (polyclonal rabbit, Dako, code A0398, 1:1000) were used. This was followed by incubation with biotinylated secondary antibodies (Dianova). For detection, alkaline phosphatase-labelled streptavidin and chromogen RED (both Dako) were employed. For detection of macrophages, sections were subjected to protein-induced epitope retrieval employing protease (Sigma) prior to incubation with anti-F4/80 (clone BM8, eBioscience, 1:800). This was followed by incubation with biotinylated rabbit anti-rat (Dako) secondary antibody. Biotin was detected using alkaline phosphatase-labelled streptavidin (Dako). For visualization of alkaline phosphatase, chromogen RED (Dako) was used. Nuclei were counterstained with hematoxylin (Merck). Negative controls were performed by omitting the primary antibody.
9
Multicolor Flow Cytometry for Lymphocyte Analysis
10
Immunofluorescent Staining of FFPE Lung Sections
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For immunofluorescent staining, formalin-fixed and paraffin-embedded (FFPE) lung sections were cut, immersed in xylene, and then hydrated in 96% alcohol and PBS. Antigens were unmasked using DakoCytomation target retrieval solution (Dako), and nonspecific binding was blocked by adding 5% (vol/vol) normal donkey serum and Fc block (BD). Avidin was used to neutralize endogenous biotin, followed by incubation with biotin (Sigma-Aldrich). Sections were then probed with anti-B220 (clone RA3-6B2; BD) and anti-CD3 (clone M-20; Santa Cruz Biotechnology) to detect B and T cells, respectively. For analysis of B cell follicles, follicles were outlined with an automated tool of the Zeiss Axioplan 2 microscope (Zeiss), and total area and average size was calculated in squared microns.
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