Anti cd45 af700

Manufactured by BD

Anti-CD45-AF700 is a fluorescently-labeled monoclonal antibody that binds to the CD45 antigen expressed on the surface of leukocytes. It is designed for use in flow cytometry applications to identify and characterize different types of blood cells.

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Lab products found in correlation

Lsrii flow cytometer, by BD (2 mentions) Anti cd4 fitc, by Thermo Fisher Scientific (2 mentions) Anti cd8a pe, by BD (2 mentions) Anti mhcii apc, by Thermo Fisher Scientific (2 mentions) Live dead stain, by Thermo Fisher Scientific (2 mentions) Anti cd19 af647, by BD (2 mentions) Anti cd11c af488, by BD (2 mentions) Anti gr1 v450, by BD (2 mentions) Anti cd3 af700, by Thermo Fisher Scientific (2 mentions) Anti siglecf pe, by BD (2 mentions) Anti cd45 ef450, by Thermo Fisher Scientific (2 mentions) Streptavidin pe, by BD (1 mentions) Anti cd31 pe, by BD (1 mentions) Clone 30 f11, by BD (1 mentions) Clone 11d4, by BD (1 mentions) Clone ebior2 60, by Thermo Fisher Scientific (1 mentions) Anti ckit apc, by Thermo Fisher Scientific (1 mentions) Anti cd41 bv421, by BioLegend (1 mentions) Anti mouse cd16 cd32, by BD (1 mentions) Ack lysing buffer, by Lonza (1 mentions)

Spelling variants of this product

Anti-CD45 (213 citations) CD45‐AF700 (2 citations)

6 protocols using anti cd45 af700

Characterization of AGM Hematopoietic Cells

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Single-cell suspensions from the AGM region were prepared by dispase/collagenase-mediated dissociation. Antibodies used for staining of cells were anti-CD45-BV450 (BD Horizon, clone 30F11, 1:100), anti-CD45-AF700 (1:100, clone 30F11, BD pharmingen), anti-VE-cadherin-AF647 or -AF488 (1:100, Clone eBioBV13, Biolegend) and biotinylated anti-VE-Cadherin (1:50, clone 11.D4.1, Pharmingen), followed by incubation with streptavidin-PE (1:600, BD Pharmingen), anti-CD43-PE (1:200, clone eBioR2/60, eBioscience), anti-cKit-APC (1:100, clone 2B8, eBioscience), anti-CD31-PE (1:200, MEC13.3, Pharmingen), anti-Sca1-V500 (1:100, clone D7, BD Bioscience), anti-CD41-BV421 (1:100, clone MWreg30, Biolegend) and anti-Ter119-PerCp-Cy5.5 (1:100, clone TER119, eBioscience). 7-Aminoactinomycin D viability staining solution was used to exclude dead cells, and gates were set using appropriate fluorescence minus one controls. Sorting was performed on a FACSAriaII using the FACSDiva software.

Souilhol C., Gonneau C., Lendinez J.G., Batsivari A., Rybtsov S., Wilson H., Morgado-Palacin L., Hills D., Taoudi S., Antonchuk J., Zhao S, & Medvinsky A. (2016). Inductive interactions mediated by interplay of asymmetric signalling underlie development of adult haematopoietic stem cells. Nature Communications, 7, 10784.

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Mouse Bone Marrow and Spleen Cell Isolation

Cited 2 times

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Bone marrow cells were collected from the femurs and tibiae of mice by flushing the opened bones with Iscove’s Modified Dulbecco’s medium (IMDM) as previously described (Dyer et al., 2008 (link)). Spleen cell suspensions in HBSS supplemented with 1% FBS and 10 mM HEPES were prepared as described (Dyer et al., 2011 (link)). Red blood cells were lysed with ACK lysing buffer (Lonza). Live/dead stain (Invitrogen) was added to the cells and non-specific antibody binding to Fc receptors was blocked with anti-mouse CD16/CD32 (BD Biosciences). For analysis of T and B cells, cell suspensions were incubated with anti-CD45-eF450 (eBioscience), anti-CD3-AF700 (eBioscience), anti-CD4-FITC (eBioscience), anti-CD8a-PE (BD), and anti-CD19-AF647 (BD) in phosphate-buffered saline with 0.1% bovine serum albumin (PBS/BSA) at 4°C for 30 min and washed with this buffer prior to analysis. For evaluation of myeloid cells, cell suspensions were incubated with anti-CD45-AF700 (BD), anti-CD11c-AF488 (BD), anti-SiglecF-PE (BD), anti-GR1-V450 (BD) and anti-MHCII-APC (eBioscience). At least 100,000 events were collected on an LSR II flow cytometer (BD Biosciences) and findings were analyzed in FlowJo 9.2.

Rice T.A., Brenner T.A., Percopo C.M., Ma M., Keicher J.D., Domachowske J.B, & Rosenberg H.F. (2016). Signaling via pattern recognition receptors NOD2 and TLR2 contributes to immunomodulatory control of lethal pneumovirus infection. Antiviral research, 132, 131-140.

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Tumor Cell Isolation and Flow Cytometry

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Tumor cell suspensions were generated by initial mechanical disaggregation/mincing. Tumor fragments were then transferred to a solution of type V collagenase (Sigma C9263, 1 mg ml⁻¹ in 1× HBSS) supplemented with soy trypsin inhibitor (Gibco, 0.1 mg ml⁻¹) and DNase I (Sigma, 0.1 mg ml⁻¹). Tumor pieces in this disaggregation buffer were transferred to a GentleMACS tube and loaded into the OctoDissociator (Miltenyi). Samples were treated with the mTDK1 program, after which 5 ml of FACS buffer (PBS 1×, 2% FBS) was added to the sample and the mix was filtered through a 100-μm mesh (BD). The resulting cell suspension was centrifuged and resuspended in FACS buffer. Cells were then treated with Fc block (BD, 1:200 dilution) incubated at 4 °C for 15 min and stained with anti-CD45 AF700 (BD, 1:400 dilution) for 30 min at 4 °C. Cells were washed and resuspended in FACS buffer supplemented with DAPI (Sigma, 1 μg ml⁻¹ final). Stained cell suspensions were then analyzed in an MA900 sorter (Sony). EGFP⁺ cells were analyzed within the CD45⁻DAPI⁻ population.

Barriga F.M., Tsanov K.M., Ho Y.J., Sohail N., Zhang A., Baslan T., Wuest A.N., Del Priore I., Meškauskaitė B., Livshits G., Alonso-Curbelo D., Simon J., Chaves-Perez A., Bar-Sagi D., Iacobuzio-Donahue C.A., Notta F., Chaligne R., Sharma R., Pe’er D, & Lowe S.W. (2022). MACHETE identifies interferon-encompassing chromosome 9p21.3 deletions as mediators of immune evasion and metastasis. Nature Cancer, 3(11), 1367-1385.

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Characterization of Lung Cell Populations

Cited 2 times

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Lung tissue was harvested and single-cell suspensions were prepared as previously described (Garcia-Crespo et al., 2013 (link); Percopo et al., 2014a (link)). Live/Dead stain (Invitrogen) was added to the cells and Ab binding to Fc receptors was blocked with anti-mouse CD16/CD32. For analysis of T and B cells, lung suspensions were stained with anti-CD45-eF450 (eBioscience), anti-CD3-AF700 (eBioscience), anti-CD4-FITC (eBioscience), anti-CD8a-PE (BD), and anti-CD19-AF647 (BD) in PBS with 0.1% BSA at 4 °C for 30 min and washed with this buffer prior to analysis. For myeloid cell analysis, cells were stained with anti-CD45-AF700 (BD), anti-CD11c-AF488 (BD), anti-SiglecF-PE (BD), anti-GR1-V450 (BD) and anti-MHCII-APC (eBioscience). Natural killer cells were characterized by staining with anti-CD45-eF450 (eBioscience), anti-CD3-AF700 (BD), and anti-CD49/DX5-PE (BD). A minimum of 100,000 events were collected on an LSR II flow cytometer (BD Biosciences) and findings were analyzed in FlowJo 9.2.

Percopo C.M., Rice T.A., Brenner T.A., Dyer K.D., Luo J.L., Kanakabandi K., Sturdevant D.E., Porcella S.F., Domachowske J.B., Keicher J.D, & Rosenberg H.F. (2015). Immunobiotic Lactobacillus administered post-exposure averts the lethal sequelae of respiratory virus infection. Antiviral research, 121, 109-119.

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Multicolor Flow Cytometry Assay for Immune Profiling

Cited 1 time

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Tumor cell suspensions (4 × 10⁶) prepared by mechanical and enzymatic dissociation^{16 (link)} were stained in 96-wells round bottom plates with live/dead staining (Blue fluorescent reactive dye, #L34962 Invitrogen) during 20 min at room temperature. For flow-cytometry analysis and sorting, Fc receptors were blocked with anti-FcR (anti-CD16 and CD32, at 5 µg/ml, Biolegend #101339). After two washes in PBS 2% FCS, cells were stained with the following antibodies (all used at 1:100): anti-CD11b-BV421 (#562605), CD64-APC (#558539), CD11c-PeCy7 (#557401), TCRβ-BV605 (#562840) and CD4-BV711 (#563050) all purchased from BD Pharmingen; anti-CD45-AF700 (#103128), Ly6C-APCCy7 (#128025), Ly6G-BV510 (#127633), F4/80 BV650 (#123149), CD206-PE (#141706), IA/IE-BV785 (#107645) all purchased from Biolegend, and CD8-PerCPef710 (#46-0081-82) purchased from eBioscience. After washing, cells were fixed in 1% PFA, stored at 4 °C, and acquired the next day on LSR II or FORTESSA (BD Bioscience). For detection of phosphorylated proteins, cell suspensions were stimulated 3 h with DMXAA 250 µg/ml, fixed immediately in PFA 4%, permeabilized with frozen methanol 90%, stained overnight with 1:100 pTBK1-PE (#13498) antibodies (purchased from Cell signaling), then washed and further stained for multicolor flow cytometry.

Guerin M.V., Regnier F., Feuillet V., Vimeux L., Weiss J.M., Bismuth G., Altan-Bonnet G., Guilbert T., Thoreau M., Finisguerra V., Donnadieu E., Trautmann A, & Bercovici N. (2019). TGFβ blocks IFNα/β release and tumor rejection in spontaneous mammary tumors. Nature Communications, 10, 4131.

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Isolation of Human Basophils by Flow Cytometry

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For human basophil sorting, cell suspensions were incubated at 4ºC with Fc-blocking reagent (Miltenyi Biotec) or blocking 2.4G2 mAb to FcγRIII/II (BD PharMingen) and then stained for 30 min with the following antibody combination: anti-CD45 AF700 (clone: HI30), anti-FcεRI FITC (clone: CRA1), antiCD123 PE (clone: 6H6), anti-HLA-DR PerCpCy5.5 (clone: L243), anti-CD19 eFluor450 (clone: HIB19), anti-IgD PE-Cy7 (clone: IA6–2). Basophils were sorted with a FACSAria II (BD Biosciences) after exclusion of dead cells through DAPI staining. The purity of cells sorted this way was consistently >95%.

Shan M., Carrillo J., Yeste A., Gutzeit C., Garzón D.S., Walland C., Pybus M., Grasset E.K., Yeiser A.J., Matthews D.B., van de Veen W., Comerma L., He B., Boonpiyathad T., Lee H., Blanco J., Osborne L.C., Siracusa M.C., Akdis M., Artis D., Mehandru S., Sampson H.A., Berin M.C., Chen K, & Cerutti A. (2018). Secreted IgD Amplifies Humoral T Helper-2 Responses by Binding Basophils Via Galectin-9 and CD44. Immunity, 49(4), 709-724.e8.

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