Pe cy7 conjugated anti cd11b

Manufactured by BD

Sourced in United States

PE-Cy7-conjugated anti-CD11b is a fluorescently-labeled antibody that binds to the CD11b cell surface antigen. It can be used to identify and isolate cells expressing CD11b, such as monocytes and granulocytes, in flow cytometry applications.

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CD11b (454 citations) Anti-CD11b (175 citations) CD11b-PE (114 citations) CD11b-PE-Cy7 (66 citations) Anti-CD11b-PE (48 citations) Anti-CD11b-PE-Cy7 (27 citations) PE-conjugated anti-CD11b (22 citations) PE-conjugated CD11b (7 citations) PE-Cy7-conjugated anti-CD11b (clone M1/70); (3 citations) PE-Cy7 conjugated rat anti-CD11b (2 citations)

8 protocols using pe cy7 conjugated anti cd11b

Bronchoalveolar Lavage Fluid Analysis

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The mice were sacrificed at days 14 and 28 after BLM administration. The mice were anaesthetised with isoflurane inhalation, and the BALF was collected by instilling 10 mL PBS into the trachea via a 25-gauge needle. The BALF was centrifuged at 800 g for 3 min at 4 °C, and then diluted in HBSS containing 2% FBS, 10 mM HEPES, and 1% penicillin/streptomycin. The BALF was then stained with PE-Cy7-conjugated anti-CD11b (BD) for counting of infiltrating macrophages.

Suto E.G., Mabuchi Y., Toyota S., Taguchi M., Naraoka Y., Itakura N., Matsuoka Y., Fujii Y., Miyasaka N, & Akazawa C. (2020). Advantage of fat-derived CD73 positive cells from multiple human tissues, prospective isolated mesenchymal stromal cells. Scientific Reports, 10, 15073.

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Comprehensive Flow Cytometry Protocol for Immune Cell Phenotyping

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The following monoclonal antibodies (mAbs) were obtained from BD Biosciences (San Jose, CA, USA): fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-Cy7- or allophycocyanin (APC)-conjugated anti-CD3ε (clone 145-2C11); FITC-, PE-Cy7, or APC-conjugated anti-CD4 (clone RM4–5); PE-conjugated anti-CD103 (clone M290); PE-Cy7-conjugated anti-GITR (clone DTA-1); PE-Cy7-conjugated anti-CD11b (clone M1/70); APC-conjugated anti-CD25 (clone PC61); PE-conjugated anti-IL10 (clone JES5-16E3); PE-Cy7-conjugated anti-CD117 (c-kit) (clone 2B8); FITC- or PE-conjugated anti-Foxp3 (clone NRRF-30); PE-conjugated anti-IL17A (clone eBio17B7). The following mAbs from Thermo Fisher Scientific were used: FITC- or APC-conjugated anti-CD19 (clone ID3); FITC- or APC-conjugated anti-Ly-6G (Gr-1) (clone RB6-8C5). FITC- or PE-conjugated anti-IL4 (clone BVD6-24G2); FITC- or APC-conjugated anti-FcεRI (clone MAR-1); PE-conjugated anti-IFNγ (clone XMG1.2). Cells were harvested and washed twice with cold 0.5% BSA-containing PBS (FACS buffer) for staining surface markers. For blocking Fc receptors, the cells were incubated with anti-CD16/CD32 mAbs (clone 2.4G2) on ice for 10 min and subsequently stained with fluorescently labeled mAbs. Flow cytometric data were acquired using a FACSCalibur flow cytometer (Becton Dickson, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).

Park H.J., Kim T.C., Park Y.H., Lee S.W., Jeon J., Park S.H., Van Kaer L, & Hong S. (2021). Repeated α-GalCer Administration Induces a Type 2 Cytokine-Biased iNKT Cell Response and Exacerbates Atopic Skin Inflammation in Vα14Tg NC/Nga Mice. Biomedicines, 9(11), 1619.

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Multiparametric Flow Cytometric Analysis of BALF Cells

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BALF samples were collected by making an incision in the trachea and lavaging the lung twice with 0.8 ml PBS, pH 7.4. Total leukocyte counts were determined using a hemacytometer.
For flow cytometric analysis, BALF cells were incubated with 2.4G2 mAb against FcγRII/III, and stained with APC-conjugated anti-CD11c (BioLegend), PE-Cy7-conjugated anti-CD11b (BD Biosciences), FITC-conjugated anti-Ly6G (clone 1A8, BD Biosciences), PerCp-Cy5.5- conjugated anti-Ly6C (eBiosciences), and PE-conjugated anti-Siglec-F (BioLegend) mAbs for myeloid cell analysis. In some experiments, cells were stained with PE-conjugated anti-active caspase 3 using a commercially available kit from BD Biosciences before surface marker staining. To differentiate early-stage apoptotic cells from the late-stage apoptotic and necrotic cells, BALF cells were stained with Fixable Viability Dye (FVD) eFluor® 780 and Annexin V PerCP-eFluor® 710 (eBiosciences), using BUV395-conjugated anti-CD11b (BD Biosciences), FITC- conjugated anti-Ly6C (BD Biosciences), BV421-conjugated anti-Ly6G (clone 1A8), PE-conjugated anti-Siglec-F and APC-conjugated anti-CD11c mAb for cell surface markers. The stained cells were analyzed on a BD LSRII-green using BD FACSDiva and FlowJo software analysis.

Bansal S., Yajjala V.K., Bauer C, & Sun K. (2018). Interleukin-1 Signaling Prevents Alveolar Macrophage Depletion during Influenza and Streptococcus pneumoniae Coinfection. Journal of immunology (Baltimore, Md. : 1950), 200(4), 1425-1433.

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Cardiac Leukocyte Characterization by Flow Cytometry

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Total cardiac cells were gated on PerCP-conjugated CD45 (BD Biosciences), and the following antibodies were used: PE-Cy7–conjugated anti-CD11b (BD Pharmingen), PE-conjugated anti-Ly6G (lymphocyte antigen 6 complex, locus G; 1A8; BD Pharmingen), FITC-conjugated anti-Ly6C (lymphocyte antigen 6 complex, locus C; Biolegend), PE-Alexa700–conjugated anti-CD3 (eBioscience) and APC-conjugated anti-F4/80 (Bio-Rad). CD45⁺/CD11b⁺ leukocytes were gated on F4/80⁻ monocytes and then further stratified by Ly6G and Ly6C expression. Monocytes were identified as CD11b^highLy6G⁻Ly6C⁺. Neutrophils were identified as CD11b⁺Ly6G^high and T cells as CD45⁺CD3⁺. The total number of cells was then normalized to LV weight. Cells were analyzed using a flow cytometer (LSR II; BD Biosciences).^{10 (link),11 (link)}

Loyer X., Zlatanova I., Devue C., Yin M., Howangyin K.Y., Klaihmon P., Guerin C.L., Kheloufi M., Vilar J., Zannis K., Fleischmann B.K., Hwang D.W., Park J., Lee H., Menasché P., Silvestre J.S, & Boulanger C.M. (2018). Intra-Cardiac Release of Extracellular Vesicles Shapes Inflammation Following Myocardial Infarction. Circulation Research, 123(1), 100-106.

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Quantification of Myeloid-Derived Suppressor Cells

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Evaluation of the MDSC percentage was accomplished with 0.5*10⁶ cells stained with FITC-conjugated anti-CD15, PE-conjugated anti-CD33, PerCP-conjugated anti-HLA-DR, a cocktail of APC-conjugated antibodies anti-CD3, -CD56, -CD19 (Lin), APC-H7-conjugated anti-CD14 and PE-Cy7-conjugated anti-CD11b (BD Biosciences). Acquisition of 100,000 events was performed in the leukocyte-gated population on FACS CANTO II and analyzed with FACS DIVA software (BD Biosciences, USA).

Sacchi A., Tumino N., Grassi G., Casetti R., Cimini E., Bordoni V., Ammassari A., Antinori A, & Agrati C. (2018). A new procedure to analyze polymorphonuclear myeloid derived suppressor cells in cryopreserved samples cells by flow cytometry. PLoS ONE, 13(8), e0202920.

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Quantification of Lung Leukocytes by Flow Cytometry

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Leukocytes from the lungs were enumerated using flow cytometry. Lungs were excised from mice, diced and digested with 40 μg/ml Liberase (Roche Diagnostics) in Iscove’s modified Dulbecco’s medium (IMDM, Sigma) and then passed through a 70-μm cell strainer (BD Bioscience) and washed with IMDM with 5% FCS. RBCs were lysed for 5 min in RBC lysis buffer (Sigma). Cell counts were performed on a Brightline hemocytometer (Hausser Scientific) with trypan blue exclusion. Cells were seeded in 96-well U-bottom plates and incubated with Fc block (BD), followed by staining with Zombie UV™ Fixable Viability kit (Biolegend) and fluorochrome-labelled antibodies (Biolegend unless otherwise specified): BUV395-cojugated anti-CD8 (BD), BV785-conjugated anti-I-A/I-E, BV711-conjugated anti-CD64, BV650-conjugated anti-CD11c, BV510-conjugated anti-CD3, BV421-conjugated anti-NK1.1, PerCpCy5.5-conjugated anti-Ly6C, FITC-conjugated anti-CD4, PECy7-conjugated anti-CD11b, PE-Texas Red-conjugated anti-F4/80, PE-conjugated anti-Siglec F, APCCy7-conjugated anti-CD19, AF700-conjugated anti-Ly6G, AF467-conjugated anti-MRP14 (BD). Cells were washed with PBS and assessed using Fortessa X20 (BD). Analysis was performed on FlowJo (Treestar).

Lin J.W., Sodenkamp J., Cunningham D., Deroost K., Tshitenge T.C., McLaughlin S., Lamb T.J., Spencer-Dene B., Hosking C., Ramesar J., Janse C.J., Graham C., O’Garra A, & Langhorne J. (2017). Signatures of malaria-associated pathology revealed by high-resolution whole-blood transcriptomics in a rodent model of malaria. Scientific Reports, 7, 41722.

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Flow Cytometry Analysis of Immune Cells

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LPMCs suspended in staining buffer (PBS containing 1 % FBS, 5 mM EDTA and 0.02 % NaN₃) were incubated with anti-CD16/32 monoclonal antibodies (mAb) for 30 min at 4 °C to block nonspecific binding. Then, cells were stained for 30 min at 4 °C with a combination of the following fluorescent mAbs: FITC-conjugated anti-Ly6G or anti-DX5 (BD Biosciences), PE-conjugated anti-CD11c (BD Biosciences), PE-Cy7-conjugated anti-CD11b (BD Biosciences), Alexa Fluor 647-conjugated anti-Ly6C (BioLegend), APC-conjugated anti-CD3ε (eBioscience) or anti-CD19 (eBioscience). Dead cells were gated out by staining with 7-aminoactinomycin D (7-AAD) (Life Technologies, Carlsbad, CA). Stained cells were acquired using FACSCalibur (BD Biosciences) or FACSAriaIII (BD Biosciences), and data were analyzed with FlowJo software (TreeStar Inc., Ashland, OR).

Tokieda S., Komori M., Ishiguro T., Iwakura Y., Takahara K, & Inaba K. (2015). Dendritic cell immunoreceptor 1 alters neutrophil responses in the development of experimental colitis. BMC Immunology, 16, 64.

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Murine Liver Leukocyte Profiling

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RAW 264.7 cell lines were obtained from ATCC and cultured in RPMI-1640 containing 10% FBS. RAW 264.7 cells were treated with TNFα monoclonal antibody (MP6-XT3) and rat IgG1 kappa isotype control. Mouse leukocytes in the liver were purified as previously described. 18 The resulting leukocytes were incubated for 30 min at room temperature with BV421-conjugated anti-CD45 (BD), PE-Cy7 conjugated anti-CD11b (BD), APC-Cy7-conjugated live/dead, APC-conjugated anti-F4/80 (BD), FITC-conjugated anti-CD86 (BD), PE-conjugated anti-CD206 (BD) and PerCP-Cy5.5-conjugated anti-Ly6G (BD). Incubation was performed in the dark. The results were analyzed using a BD FACSCalibur flow cytometer and FlowJo software (Treestar, Ashland, OR, USA).

Yin Y., Kong D., He K, & Xia Q. (2022). Aurora kinase A regulates liver regeneration through macrophages polarization and Wnt/β-catenin signalling. Liver international : official journal of the International Association for the Study of the Liver, 42(2).

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