Anti cd45r b220 clone ra3 6b2

Manufactured by BD

Sourced in Denmark, United States

The Anti-CD45R/B220 (clone RA3-6B2) is a laboratory reagent that can be used to detect the CD45R/B220 antigen. CD45R/B220 is a protein expressed on the surface of B lymphocytes. This reagent can be used in various immunological techniques such as flow cytometry to identify and characterize B cells.

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Lab products found in correlation

Streptavidin pe, by Thermo Fisher Scientific (1 mentions) Anti cd45.2 clone 104, by Thermo Fisher Scientific (1 mentions) Anti fcεriα clone mar 1, by Thermo Fisher Scientific (1 mentions) Bioline isolate 2 rna micro kit, by Meridian Bioscience (1 mentions) Anti cd3 clone 2c11, by BD (1 mentions) Anti cd11c clone n418, by BioLegend (1 mentions) Dispase 2, by Roche (1 mentions) Collagenase d, by Roche (1 mentions) Dnase 1, by Roche (1 mentions) Anti cd16 cd32 fc block clone 2.4g2, by BD (1 mentions) Fixable viability dye efluor 506, by Thermo Fisher Scientific (1 mentions) Aperio scanscope cs2 system, by Leica Biosystems (1 mentions) Anti cd11b alexa647, by BioLegend (1 mentions) Anti rabbit alexa488, by Thermo Fisher Scientific (1 mentions) Anti cd34 clone mec14, by BioLegend (1 mentions) Anti cd3 clone sp7, by Abcam (1 mentions) Anti goat alexa 647, by Thermo Fisher Scientific (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Rabbit polyclonal anti cd3, by Agilent Technologies (1 mentions) Anti lyve 1, by Abcam (1 mentions)

6 protocols using anti cd45r b220 clone ra3 6b2

Isolation and Characterization of Murine Mast Cells

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Ears were harvested and separated into dorsal and ventral halves using forceps. For epidermal removal, the skin was incubated epidermis-down in 1.2 mg/ml Dispase II (Roche) at 37°C. After an hour, the epidermal layer was peeled off the dermis. To release cells, skin were torn into small fragments and digested for 1 h in 1 mg/ml collagenase D, 0.5 mg/ml type 2 hyaluronidase and 20 ug/ml Dnase 1 (all from Roche) at 37°C. Tissues were passed through a cell strainer and washed in PBS containing 3% FBS. Isolated cells were then stained for flow cytometry or cell sorting using anti-CD3 (clone 2C11, 1.0 µg/ml), anti-CD45R/B220 (clone RA3-6B2, 1.0 µg/ml), and anti-CD117 (cKit clone 2B8, 1.25 µg/ml) antibodies from BD Pharmingen, and anti-CD45.2 (clone 104, 0.5 µg/ml), anti-FcεRIα (clone MAR-1, 0.5 µg/ml) antibodies and streptavidin PE (0.4 µg/ml) from eBioscience, and anti-CD11c (clone N418, 2.5 µg/ml) from BioLegend. MCs were gated as CD45.2⁺, CD3⁻, B220⁻, CD11c⁻, cKit⁺ and FcεR1α⁺. For mRNA isolation, sorted MCs were directly collected into lysis buffer (Bioline ISOLATE II RNA Micro Kit).

Bergot A.S., Ford N., Leggatt G.R., Wells J.W., Frazer I.H, & Grimbaldeston M.A. (2014). HPV16-E7 Expression in Squamous Epithelium Creates a Local Immune Suppressive Environment via CCL2- and CCL5- Mediated Recruitment of Mast Cells. PLoS Pathogens, 10(10), e1004466.

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Multiparameter Flow Cytometric Analysis of Spleen Cells

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Spleen cells were surface stained with Fixable Viability Dye eFluor506 (eBioscience), anti-CD16/CD32 Fc block (clone 2.4G2; BD Biosciences), anti-CD3-PE-Cy5 (145-2C11; Tonbo Biosciences), anti-CD45R/B220 (clone RA3-6B2; BD Biosciences), anti-CD21/CD35-eFluor450 (clone 4E3; eBioscience), and anti-CD23-PE-Cy7 (clone B3B4,eBioscience) for 20 min.

Demeyer A., Skordos I., Driege Y., Kreike M., Hochepied T., Baens M., Staal J, & Beyaert R. (2019). MALT1 Proteolytic Activity Suppresses Autoimmunity in a T Cell Intrinsic Manner. Frontiers in Immunology, 10, 1898.

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Immunohistochemical and Immunofluorescence Analysis

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At the time of autopsy, different organs for each mouse were sampled and either fixed in zinc–formalin or 4% paraformaldehyde and processed as described in Appendix Supplementary Materials and Methods. Immunohistochemical staining was performed utilizing the following antibodies: anti‐F4/80 (clone A3‐1, AbD Serotec); anti‐RFP (rabbit polyclonal, ab62341 AbCam); anti‐CD34 (clone MEC14.7, Biolegend); anti‐Ki67 (clone SP6, Neomarkers); anti‐CD3 (clone SP7, AbCam); and anti‐CD45R/B220 (clone RA3‐6B2, BD Pharmingen). All images were acquired using the Aperio Scanscope CS2 system (Leica Biosystems). Immunofluorescence staining was performed utilizing the following antibodies: anti‐GFP (rabbit polyclonal, A11122 Invitrogen) + anti‐rabbit Alexa 488 (Invitrogen); anti‐MMR (goat polyclonal, AF2535 R&D Systems) + anti‐goat Alexa 647 (Invitrogen); anti‐F4/80‐PE (clone A3‐1, AbD Serotec); anti‐CD11b‐Alexa 647 (clone M1/70; Biolegend); and Hoechst 33342 (Invitrogen). Confocal images were acquired using a Leica TCS SP2 or SP8 confocal system (Leica Microsystems) that are available at the SRSI Advanced Light and Electron Microscopy BioImaging Center (ALEMBIC).

Catarinella M., Monestiroli A., Escobar G., Fiocchi A., Tran N.L., Aiolfi R., Marra P., Esposito A., Cipriani F., Aldrighetti L., Iannacone M., Naldini L., Guidotti L.G, & Sitia G. (2016). IFNα gene/cell therapy curbs colorectal cancer colonization of the liver by acting on the hepatic microenvironment. EMBO Molecular Medicine, 8(2), 155-170.

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Histological and Immunohistochemical Analysis of Colon Cancer

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Paraffin-embedded sections (3-μm-thick) of the colon wall were prepared and stained with H&E for histological analysis. Cross-sections of colon wall were routinely cut in Apc^Min/+ mice and AOM/DSS-treated mice. Horizontal sections of colon wall were prepared in AOM alone-treated mice and the number of intramucosal microadenomas was counted on the H&E-stained section.23 (link) Immunohistochemical analyses were performed as described previously using the following primary antibodies: rabbit polyclonal anti-IDO (provided by K. Saito, Kyoto University),24 (link) rabbit polyclonal anti-Foxp3 (Abcam, Cambridge, UK), rabbit polyclonal anti-CD3 (DAKO, Glostrup, Denmark) and rat monoclonal anti-CD45R/B220 (clone RA3-6B2; BD Biosciences, San Jose, CA, USA). Double immunostaining for IDO and CD11c was performed as described previously with a slight modification.16 (link) Ethical approval for use of archival tissues of human CRC was provided by the Ethics Committee of Gifu University School of Medicine.

Takamatsu M., Hirata A., Ohtaki H., Hoshi M., Ando T., Ito H., Hatano Y., Tomita H., Kuno T., Saito K., Seishima M, & Hara A. (2015). Inhibition of indoleamine 2,3-dioxygenase 1 expression alters immune response in colon tumor microenvironment in mice. Cancer Science, 106(8), 1008-1015.

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Histological Analysis of Granulomas

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Granulomas were excised from the dorsal skin region one week after the third injection (i.e., 13 weeks of age) and fixed in 10% formalin solution overnight at room temperature. Fixed tissues were processed to paraffin blocks by the Molecular Pathology Core at the University of Florida. Paraffin sections (4 μm) were stained with H&E, or by immunohistochemistry with the following primary antibodies: anti- CD45R/B220, clone RA3-6B2 (BD Biosciences, San Jose, CA, USA), anti- CD3, clone CD3-12 (Serotec, Raleigh, NC, USA), anti- F4/80, clone BM8 (Caltag Medsystems, Buckingham, UK), or anti- LYVE-1 (Lymphatic vessel endothelial hyaluronan receptor-1; Abcam, Cambridge, MA, USA). Deparaffinized sections were hydrated then blocked in normal goat serum followed by incubation in primary antibodies for one hour at room temperature. Following washes, sections were incubated with goat anti-rabbit HRP-polymer labeled antibody (Mach2 HRP, Biocare, Concord, CA, USA) for 30 min at room temperature followed by horseradish peroxidase- DAB and hematoxylin counterstain.

Yoon Y.M., Lewis J.S., Carstens M.R., Campbell-Thompson M., Wasserfall C.H., Atkinson M.A, & Keselowsky B.G. (2015). A combination hydrogel microparticle-based vaccine prevents type 1 diabetes in non-obese diabetic mice. Scientific Reports, 5, 13155.

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Quantifying BrdU-Labeled Hematopoietic Stem Cells

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For BrdU labeling, C57BL/6 mice were intraperitoneally injected with 1 mg BrdU (10 mg/mL in PBS) 2 days after myocardial infarction and euthanized 24 hours later. Bone marrow was flushed out of the femur with PBS, filtered through a 70-μm cell strainer, incubated with lysis buffer for 30 seconds, and washed with FACS buffer. Cells were stained with APC/Cy7 anti-CD48 (clone HM48-1, 1:80; BioLegend), PerCP/Cy5.5 anti-CD150 (clone TC15-12F12.2, 1:160; BioLegend), PE anti-CD117 (c-Kit, clone 2B8, 1:400; BioLegend), anti–Sca-1 (Ly6-A/E, clone E13-161.7, 1:160; BioLegend), and a lineage cocktail (1:10; BD Biosciences) containing anti-CD3ε (clone 500A2), anti-CD11b (clone M1/70), anti-CD45R/B220 (clone RA3-6B2), anti-Ly76 (clone TER-119), and anti-Ly6G/Ly6C (clone RB6-8C5). BrdU staining was performed with the use of the BrdU staining kit (BD FITC BrdU Flow Kit; Fisher Scientific), following the manufacturer’s instructions. LSK cells were identified as Lin⁻, Sca-1⁺, and c-Kit⁺.

Maier A., Toner Y.C., Munitz J., Sullivan N.A., Sakurai K., Meerwaldt A.E., Brechbühl E.E., Prévot G., van Elsas Y., Maas R.J., Ranzenigo A., Soultanidis G., Rashidian M., Pérez-Medina C., Heo G.S., Gropler R.J., Liu Y., Reiner T., Nahrendorf M., Swirski F.K., Strijkers G.J., Teunissen A.J., Calcagno C., Fayad Z.A., Mulder W.J, & van Leent M.M. (2023). Multiparametric Immunoimaging Maps Inflammatory Signatures in Murine Myocardial Infarction Models. JACC: Basic to Translational Science, 8(7), 801-816.

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