Pe labeled anti cd11b

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PE-labeled anti-CD11b is a monoclonal antibody that binds to the CD11b cell surface antigen. CD11b is a component of the integrin Mac-1 (CD11b/CD18) and is expressed on the surface of various immune cells, including monocytes, macrophages, and neutrophils. The PE (phycoerythrin) fluorescent label allows for the detection and analysis of CD11b-expressing cells using flow cytometry.

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CD11b (457 citations) Anti-CD11b (179 citations) CD11b-PE (81 citations) Anti-CD11b-PE (52 citations) PE-labeled CD11b (2 citations) PE-labeled anti-CD11b (clone M1/70) (2 citations)

6 protocols using pe labeled anti cd11b

Cell Differentiation Assay with FTO Knockdown

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For in vitro cell differentiation assay, FTO knockdown and control cells were incubated with 250 ng/μl Ara-C (#S1648, Selleck) for 48 h, cells were harvested and washed with chilled PBS and stained with PE-labeled anti-CD11b (#301306, BioLegend, California, USA) and APC-labeled anti-CD14 (#367118, Biolegend) for flow cytometry analysis. The fluorescence intensity was detected using CANTO PLUS (BD Biosciences, New Jersey, USA) and analyzed with the FlowJo.

Zhang Z.W., Zhao X.S., Guo H, & Huang X.J. (2023). The role of m6A demethylase FTO in chemotherapy resistance mediating acute myeloid leukemia relapse. Cell Death Discovery, 9, 225.

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Isolation of Murine Myeloid-Derived Suppressor Cells

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Myeloid-derived suppressor cells were isolated from bone marrow cells by magnetic bead sorting using the MACS system (Miltenyi Biotech, Germany). Bone marrow cells were obtained from the femurs and tibias of female Balb/c mice using a syringe. According to the manufacturer’s instructions, positive selection of Gr-1^highLy-6G⁺ was performed with LS columns. After the first depletion of Ly-6G⁺ cells using LS columns, Gr-1^dimLy-6G^- cells were enriched, and subsequent positive selection of Gr-1⁺ cells was performed using MS columns. The isolated cells were identified by flow cytometry using PerCP/cy5.5-labeled anti-CD45, PE-labeled anti-CD11b, and APC-labeled anti-Gr-1 (Biolegend, USA).

Ouyang L., Chang W., Fang B., Qin J., Qu X, & Cheng F. (2016). Estrogen-induced SDF-1α production promotes the progression of ER-negative breast cancer via the accumulation of MDSCs in the tumor microenvironment. Scientific Reports, 6, 39541.

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Neutrophil Exocytosis Pathway Analysis

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Exocytosis of the primary/secondary granules within the human neutrophils was analyzed by monitoring the cell surface proteins CD11b, CD15, and CD63.28 (link) Briefly, human neutrophils were co-cultured with MDRAB in the absence or presence of 8 μg/mL TGC and harvested after 4 h. The harvested cells were washed with phosphate buffered saline (PBS) (Nacalai Tesque) and enumerated. To analyze the expression of the cell surface proteins, human neutrophils were stained with specific monoclonal antibodies (mAbs) against the cell surface markers for 30 min at 4 °C. The mAbs included phycoerythrin (PE)-labeled anti-CD11b, allophycocyanin (APC)-labeled anti-CD63, and APC-Cy-7-labeled CD15 (BioLegend, San Diego, CA, USA). The cells were washed twice with staining buffer (BD Biosciences, San Jose, CA, USA) and subsequently fixed with the BD Cytofix™ Fixation Buffer (BD Biosciences). The stained cells were analyzed using a FACSCant II flow cytometer that was equipped with the FACS Diva software (BD Biosciences). All flow cytometry data were analyzed using the FlowJo v10.8.0 software (BD Biosciences).

Sato Y., Hatayama N., Ubagai T., Tansho-Nagakawa S., Ono Y, & Yoshino Y. (2022). Tigecycline Suppresses the Virulence Factors of Multidrug-Resistant Acinetobacter baumannii Allowing Human Neutrophils to Act. Infection and Drug Resistance, 15, 3357-3368.

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Monocytic Cell Differentiation Assay

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After counting with trypan blue exclusion, 20000–50000 cells were loaded and cytospins were prepared at 1000 rpm for 5 min. Slides were let dry and stained with StainRITE® Wright-Giemsa Stain Solution (Polysciences, Warrington, PA) and mounted with Poly-mount (Polysciences).
For flow cytometric analysis, MM6, NB4 or U937 cells with different treatments were harvested and washed with chilled PBS, followed by staining with PE-labeled anti-CD11b (101208, BioLegend) and APC-labeled anti-CD14 (17-0149-41, eBioscience) antibodies for 25 minutes. Cells were then fixed and analyzed on a BD LSRFortessa or FACSAria III analyzer (BD Biosciences).

Weng H., Huang H., Wu H., Qin X., Zhao B.S., Dong L., Shi H., Skibbe J., Shen C., Hu C., Sheng Y., Wang Y., Wunderlich M., Zhang B., Dore L.C., Su R., Deng X., Ferchen K., Li C., Sun M., Lu Z., Jiang X., Marcucci G., Mulloy J., Yang J., Qian Z., Wei M., He C, & Chen J. (2017). METTL14 Inhibits Hematopoietic Stem/Progenitor Differentiation and Promotes Leukemogenesis via mRNA m6A Modification. Cell stem cell, 22(2), 191-205.e9.

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Flow Cytometry Analysis of Dendritic Cell Phenotype

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For flow cytometry analysis of DC phenotype we used the following monoclonal antibodies (mAbs): FITC-labeled anti-CD1a (clone: HI149), anti-CD1c (clone: L161), anti-CD14 (clone: 63D3), anti-CD16 (clone: 3G8), and anti-HLA I (clone: W6/32) (all from Biolegend, CA, USA); Alexa Fluor 488-labeled anti-CD4 (clone: RPA-T4) (Biolegend); PE-labeled anti-CD11b (clone: ICRF44), anti-CD11c (clone: 3.9), anti-DC-SIGN (clone: 9E9A8), anti-CD80 (clone: 2D10), anti-CD83 (clone: HB15e), anti-CD86 (clone: BU63) (all from Biolegend), and anti-HLA II (clone: AC122), anti-ILT-3 (clone: REA141), anti-ILT4 (clone: REA184), anti-PDL-1 (clone: REA1197), and anti-FasL (clone: NOK-1) (all from Miltenyi Biotec). Briefly, variously treated DCs were harvested and collected by centrifugation. Antibody was added and the cells were incubated for 15 min in the dark, then washed twice and resuspended in 2% paraformaldehyde (PFA). Samples were analyzed on a FACSCalibur system (Becton Dickinson, Inc.). Data was analyzed with the CellQuest software (BD biosciences).
Endocytosis was monitored by flow cytometry after incubation of DCs with FITC-dextran (1 mg/ml), either on ice or at room temperature, for 1 h. The cells were then washed twice with DPBS and re-suspended in 2% PFA for future analysis.

Švajger U, & Rožman P.J. (2019). Synergistic Effects of Interferon-γ and Vitamin D3 Signaling in Induction of ILT-3highPDL-1high Tolerogenic Dendritic Cells. Frontiers in Immunology, 10, 2627.

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Immunophenotyping of Mouse and Human Leukocytes

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(Bajt et al., 2001 (link); Daley et al., 2008 (link)). 5 μg Fc receptor (FcR) blocking antibody (BioLegend, San Diego, CA) diluted in 100 μL 0.1% BSA in PBS was added to 100 μL non-parenchymal cell suspension for 20 minutes on ice (mouse only). To 50 μL heparinized whole blood (mouse or human) or 200 μL FcR-blocked hepatic non-parenchymal cells (mouse only), saturating concentrations of PE-Cy5-labeled-anti-Gr-1 [mouse only (BioLegend)] and PE-labeled-anti-CD11b [human or mouse (BioLegend)] diluted in 0.1% BSA in PBS were added. Tubes were incubated in the dark, on ice for 30 minutes. After washing with 0.1% BSA, red blood cells were lysed using RBC lysing solution (0.155 M NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA). Pellets were washed three times with 0.1% BSA then fixed with 2.5% buffered formalin. Samples were measured on the FACSCalibur (BD, Franklin Lakes, NJ). Mouse neutrophils were gated on the Gr-1^high cells, which are the Ly6G-positive population and identified as neutrophils by cell morphology (Daley et al., 2008 (link)). Other leukocytes are either Gr-1^intermediate (mainly monocytes and eosinophils) or Gr-1^negative (monocytes and lymphocytes) (Daley et al., 2008 (link)). Human granulocytes (primarily comprised of neutrophils) were gated to exclude monocytes and lymphocytes by forward- and side-scatter characteristics. The data were analyzed using the BD FACSDiva 6.0 software.

Williams C.D., Bajt M.L., Sharpe M.R., McGill M.R., Farhood A, & Jaeschke H. (2014). Neutrophil Activation During Acetaminophen Hepatotoxicity and Repair in Mice and Humans. Toxicology and applied pharmacology, 275(2), 122-133.

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