Pe cd11b antibody

Manufactured by BioLegend

Sourced in United States

The PE-CD11b antibody is a flow cytometry reagent that binds to the CD11b cell surface antigen. CD11b is a component of the integrin complex Mac-1 (CD11b/CD18) and is expressed on the surface of various immune cells, including monocytes, macrophages, and granulocytes. The PE-CD11b antibody can be used for the identification and analysis of cells expressing CD11b.

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9 protocols using pe cd11b antibody

Isolation and Analysis of Tumor-Associated Monocytes/Macrophages

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Monocytes or macrophages co-cultured with tumor cells were sorted by FACS (BD FASC Canto™), using CD14-FITC (Biolegend, 325603), CD11b-PE antibodies (Biolegend, 982606) or pre-labeled fluorescent dyes. At the end of each isolation a sorting purity check was performed. The sorted cells were cultured with the corresponding medium.
For the cytofluorimetric analysis assay, the sorted monocytes or macrophages were harvested and re-suspended in PBS with 1% BSA. Fc receptors blocker was used to reduce the non-specific immunofluorescent staining by incubating samples for 20min on ice. 1x10⁶ cells were stained in a final volume of 100 μL using the following antibodies at 1:100 dilutions: CD11b-FITC (Biolegend, 301330), CD206-APC (Biolegend, 321109), SIGLEC7-APC (Biolegend,339205), SIGLEC9-PE (R&D, FAB1139P-025), CD14-APC (Biolegend, 301808), CD16-FITC (Biolegend, 302005).
For the in vitro phagocytosis assay by flow cytometry, we constructed a co-culture system in which tumor cells were co-cultured with macrophages in a 1:3-5 ratio. After 24 hours, the cells were harvested and analyzed by flow cytometry.

Tian L., Li H., Zhao P., Liu Y., Lu Y., Zhong R., Jin Y., Tan T, & Cheng Y. (2023). C-Myc-induced hypersialylation of small cell lung cancer facilitates pro-tumoral phenotypes of macrophages. iScience, 26(10), 107771.

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Microglia Regulation by MG in LPS-Induced Inflammation

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MG (purity >98%) was purchased from SanHerb Bioscience (Chengdu, Sichuan, China). Carboxyl methyl cellulose sodium (CMC-Na) was obtained from Aladdin (Shanghai, China). Lipopolysaccharide (LPS, from the Gram-negative bacterium E. coli 055:B5) and methyllycaconitine (MLA, α7nAChR specific inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) were bought from Hyclone (Logan City, UT, USA). Fetal bovine serum (FBS) was purchased from Gibco (CA, USA). Lymphocyte separation solution was bought from Solarbio (Beijing, China). Anti-rat CD4-PE, IFN-γ-FITC, CD86-FITC and CD11b-PE antibodies were purchased from BioLegend (San Diego, CA, USA). Anti-rat p65, ac-p65, p-p65, α7nAChR and SIRT1 antibodies were the products of Affinity Biosciences (Changzhou, Jiangsu, China). Biotin-conjugated secondary antibodies were supplied by Beyotime Biotech (Nantong, Jiangsu, China). Acetylcholinesterase (AChE) assay kit and Coenzyme I NAD (H) assay kit were supplied by JianCheng Bioengineering Institute (Nanjing, Jiangsu, China).

Chen W.G., Zhang S.S., Pan S., Wang Z.F., Xu J.Y., Sheng X.H., Yin Q, & Wu Y.J. (2022). α-Mangostin Treats Early-Stage Adjuvant-Induced Arthritis of Rat by Regulating the CAP-SIRT1 Pathway in Macrophages. Drug Design, Development and Therapy, 16, 509-520.

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Flow Cytometry of Macrophages

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SVF cells or BMDMs were blocked with TrueStain fcX (BioLegend) for 15 min on ice in PBS containing 0.5% FBS, 2 mM EDTA. SVF cells were incubated with F4/80-APC antibody (BioLegend) or rat isotype-APC control antibody (BioLegend) for 30 min on ice. BMDMs were incubated with both F4/80-APC antibody (BioLegend) and CD11-b-PE antibody (BioLegend) for 30 min on ice. Both rat isotype-APC and rat isotype-PE control antibodies (BioLegend) were used in BMDMs as controls. The cells were washed and subjected to flow cytometry analysis using a DxP10 FACSort flow cytometer (BD Biosciences, San Jose, CA, USA). The data were analyzed with FlowJo (BD Biosciences).

Liu B., Li X., Yu H., Shi X., Zhou Y., Alvarez S., Naldrett M.J., Kachman S.D., Ro S.H., Sun X., Chung S., Jing L, & Yu J. (2021). Therapeutic potential of garlic chive-derived vesicle-like nanoparticles in NLRP3 inflammasome-mediated inflammatory diseases. Theranostics, 11(19), 9311-9330.

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Annexin V-PI Apoptosis and Cell Cycle Analysis

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Staining of apoptotic cells was performed with the Annexin V-PI Apoptosis Detection Kit (BD Biosciences, CA, USA) according to the manufacturers' instructions. For the cell cycle analysis, following treatment with Nec-1 for 48h, the cells were washed in PBS and fixed in 70% ethanol overnight at -20 °C. Following this the cells were incubated with PI and RNase for 30 min at 37 °C after being washed with PBS. Before flow cytometry, the cells were stained with the CD11b-PE antibody (BioLegend, no. 301306) for the differentiation analysis.

Dan W., Zhong L., Zhang Z., Wan P., Lu Y., Wang X., Liu Z., Chu X, & Liu B. (2022). RIP1-dependent Apoptosis and Differentiation Regulated by Skp2 and Akt/GSK3β in Acute Myeloid Leukemia. International Journal of Medical Sciences, 19(3), 525-536.

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Flow Cytometric Analysis of CD11b Knockdown

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Flow cytometric analysis was performed on a Fortessa or FACSCalibur FACS machine (BD). Standard staining was performed. In brief, cells were washed three times with FACS buffer and blocked using Fc-block CD16/32 antibody (BioLegend) for 15 min, followed by incubation with fluorescently labeled antibody for 30 min. Analysis was performed using FlowJo software (version 9.2; Tree Star). PBMCs were isolated by Ficoll centrifugation. The antibodies used were: CD11b-PE antibody (BioLegend), CD162–Alexa Fluor 647 (BD), CD62L-FITC and CD18-PE (eBioscience), and CD54-APC, CD14-Pe/Cy7, CD49d-FITC, CD11a-PE, CD11b–eFluor 450 (all BioLegend). Sterile FACS to maintain CD11b knockdown in CD11b^kd BV2 cell lines was performed after staining of CR3^kd and control cells with CD11b-PE antibody, as described in the previous paragraph. Cells were sorted on a FACSAriaIII cell sorter (BD) for CD11b^low-expressing cells, defined by CD11b expression of <80% of control cells. Then, cells were cultured as described in the Cell lines section.

Czirr E., Castello N.A., Mosher K.I., Castellano J.M., Hinkson I.V., Lucin K.M., Baeza-Raja B., Ryu J.K., Li L., Farina S.N., Belichenko N.P., Longo F.M., Akassoglou K., Britschgi M., Cirrito J.R, & Wyss-Coray T. (2017). Microglial complement receptor 3 regulates brain Aβ levels through secreted proteolytic activity. The Journal of Experimental Medicine, 214(4), 1081-1092.

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ATRA-Induced Granulocytic Differentiation

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Granulocytic differentiation in NB4 cells was induced with ATRA (Sigma-Aldrich). Morphology was evaluated by conventional light-field microscopy to examine May-Grünwald-Giemsa-stained cytospins using an Olympus BX51 (Japan) optical microscope, and the CD11b levels were determined using the PE-CD11b antibody (BioLegend, #301305) and a FACSAria Flow Cytometer (BD Biosciences). Real-time PCR and western blots were used to analyze the expression level of DHX15 in NB4 cells with and without ATRA treatment.

Pan L., Li Y., Zhang H.Y., Zheng Y., Liu X.L., Hu Z., Wang Y., Wang J., Cai Y.H., Liu Q., Chen W.L., Guo Y., Huang Y.M., Qian F., Jin L., Wang J, & Wang S.Y. (2017). DHX15 is associated with poor prognosis in acute myeloid leukemia (AML) and regulates cell apoptosis via the NF-kB signaling pathway. Oncotarget, 8(52), 89643-89654.

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Flow Cytometric Analysis of Immune Markers

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For flow cytometric analyses of cell surface CD11b and CD86, cells were washed with PBS then stained with PE-CD11b antibody (Biolegend, #101207) or FITC-CD86 antibody (Biolegend, #374203) at room temperature for 15 minutes and then washed twice with cold PBS for flow cytometric analysis. For EdU (Life Technologies, #C10634), cell senescence (Thermo Scientific #C10840) and Annexin-V cell apoptosis (Life Technologies, #A23204) analyses, cells were prepared following product instructions and then analyzed by flow cytometry. Flow cytometry was performed on BD LSRFortessa (BD Biosciences) and analyzed with FlowJo software (Treestar). Fluorescence activated cell sorting (FACS) of AML cells was performed by MoFlo Astrios (Beckman) or BD Jazz (BD Biosciences) according to the manufacturer’s instructions.

Yan F., Li J., Milosevic J., Petroni R., Liu S., Shi Z., Yuan S., Reynaga J.M., Qi Y., Rico J., Yu S., Liu Y., Rokudai S., Palmisiano N., Meyer S.E., Sung P.J., Wan L., Lan F., Garcia B.A., Stanger B.Z., Sykes D.B, & Andrés Blanco M. (2022). KAT6A and ENL form an epigenetic transcriptional control module to drive critical leukemogenic gene expression programs. Cancer discovery, 12(3), 792-811.

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Comprehensive Tumor Immune Profiling

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Melanoma tumor tissues were prepared as single cell suspensions, blocked with anti-CD16/CD32, and the dead cells were removed with a Zombie Red Fixable Viability Kit. Single cells were stained using APCCY7-CD45 antibody (BioLegend, CAT#:103116, USA), APC-CD3 antibody (BioLegend, CAT#:100236, USA), PE-Cy5.5-CD4 antibody (BioLegend, CAT#:100434 USA), PE-Cy7-CD8 antibody (BioLegend, CAT#:100722, USA), PE/Dazzle 594-CD274 antibody (BioLegend, CAT#:124324, USA), APC-F4/80 antibody (BioLegend, CAT#:123116, USA) and PE-CD11B antibody (BioLegend, CAT#:101208, USA) for 30 min. After fixation and permeabilization (eBioscience, USA), intracellular staining was performed using FITC-Granzyme B antibody (BioLegend, CAT#:372206, USA), BV711-IFNγ antibody (BioLegend, CAT#:505836, USA) and PE-Cy7-CD206 antibody (BioLegend, CAT#:141720, USA). The antibodies used in animal flow cytometry are from biolegend, Inc. The stained cells were analyzed by FACS (Cytek, USA) and Flow Jo software.

Liu N., Zhang J., Yan M., Chen L., Wu J., Tao Q., Yan B., Chen X, & Peng C. (2023). Supplementation with α-ketoglutarate improved the efficacy of anti-PD1 melanoma treatment through epigenetic modulation of PD-L1. Cell Death & Disease, 14(2), 170.

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Multiparametric Flow Cytometry Analysis

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The cells or tumor tissues were prepared as single-cell suspensions; 100 μl of prepared Zombie Aqua Fixable Viability Kit (anti-BV510, BioLegend, USA) was added and incubated for 10 to 15 min at room temperature; configured CD16/32 (BioLegend) was added, incubated for 15 min at 4 °C, and protected from light; and 100 μl of prepared surface antibody [APCCY7-CD45 antibody, APC-CD3 antibody, PE-Cy5.5-CD4 antibody, PE-Cy7-CD8 antibody, PE-NK1.1, PE-Cy5.5-CD25, PE-Cy5.5-Gr-1, APC-F4/80 antibody, and PE-CD11B antibody (BioLegend)] was added and incubated for 30 min at 4 °C. To label intracellular molecules, cells were incubated for 30 min from light after fixation and permeabilization (eBioscience, USA); 100 μl of the prepared intracellular antibody [FITC-Granzyme B antibody (BioLegend), PE-FOXP3 antibody (eBioscience), and BV711-IFN-γ antibody (BioLegend)] was added and incubated for 30 min at 4 °C. The stained cells were analyzed by FACS LSRFORTESSA (BD, USA), and the data were analyzed by FLOWJO software.

Liu N., Chen L., Yan M., Tao Q., Wu J., Chen J., Chen X., Zhang W, & Peng C. (2023). Eubacterium rectale Improves the Efficacy of Anti-PD1 Immunotherapy in Melanoma via l-Serine-Mediated NK Cell Activation. Research, 6, 0127.

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