Fitc anti mouse human cd11b antibody

Manufactured by BioLegend

Sourced in United States, Germany

The FITC anti-mouse/human CD11b antibody is a fluorescently labeled antibody that binds to the CD11b cell surface antigen, which is expressed on myeloid cells, including monocytes, macrophages, and granulocytes. This antibody can be used for the identification and analysis of CD11b-positive cells in various applications, such as flow cytometry.

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9 protocols using fitc anti mouse human cd11b antibody

Multiparametric flow cytometry for immune cell profiling

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Single-cell suspensions were prepared by using the multi-tissue dissociation kit 2 (Miltenyi Biotec). For cytometric analyses, the cells were incubated with a mixture of antibodies at 4°C for 20 min. The antibodies used in the present study are listed: FITC anti-mouse/human CD11b Antibody (BioLegend) (Cat#101206), PerCP/Cyanine5.5 anti-mouse CD45 Antibody (BioLegend) (Cat#103132), APC anti-mouse Ly-6G Antibody (BioLegend) (Cat#127614), Zombie Aqua Fixable Viability Kit (BioLegend) (Cat# 423102). The obtained results were expressed as the percent or cell number per microgram of tissue. Flow cytometric analysis and cell sorting were performed on an LSRFORTESSA and FACS Aria instruments (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star).

Sun X., Chen H., Gao R., Huang Y., Qu Y., Yang H., Wei X., Hu S., Zhang J., Wang P., Zou Y., Hu K., Ge J, & Sun A. (2023). Mitochondrial transplantation ameliorates doxorubicin-induced cardiac dysfunction via activating glutamine metabolism. iScience, 26(10), 107790.

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Profiling Tumor Immune Landscape by Flow Cytometry

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CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells and CD3⁺CD4⁺FOXP3⁺ T cells, CD11c ⁺CD86⁺CD80⁺ dendritic cells as well as CD45⁺CD11b⁺F4/80⁺CD206⁺ M2 like macrophage and CD45⁺CD11b⁺F4/80⁺CD86⁺ M1 like macrophage in the tumor tissues or spleen were isolated and analyzed using flow cytometry. The antibodies involved in the experiment include FITC anti-mouse CD11c Antibody (Biolegend, Cat# 117306, Clone No. N418), PE anti-mouse CD86 Antibody (Biolegend, Cat# 105007, Clone No.GL-1), APC anti-mouse CD80 Antibody (Biolegend, Cat# 104714, Clone No.16-10A1), FITC anti-mouse CD3 (Biolegend, Cat# 100203, Clone No.17A2), PE anti-mouse CD4 (Biolegend, Cat# 100407, Clone No.GK1.5), APCanti-mouseCD8a (Biolegend, Cat# 100712, Clone No.53-6.7), AlexaFluor488 anti-mouse FOXP3 (Biolegend, Cat# 320012, Clone No.150D), FITC anti-mouse/human CD11b Antibody (Biolegend, Cat# 101205, Clone No. M1/70), APC anti-mouse CD45 (Biolegend, Cat# 103112, Clone No.30-F11), PerCP anti-mouse F4/80 Antibody (Biolegend, Cat# 123126, Clone No.BM8) and PE/Cyanine7 anti-mouse CD206 (Bioegend, Cat# 141720, Clone No.C068C2). The antibody concentration was 1:100 diluted with PBS.
IL-2, IL-12p70, TNF-α, and IFN-γ in primary tumors were also examined with ELISA kits. And tumors were stained for immunofluorescence of CD3⁺CD4⁺ and CD3⁺CD8⁺ proliferated CTLs in 4T1 tumor tissue slices of the primary tumor.

Yu J., Zhou B., Zhang S., Yin H., Sun L., Pu Y., Zhou B., Sun Y., Li X., Fang Y., Wang L., Zhao C., Du D., Zhang Y, & Xu H. (2022). Design of a self-driven probiotic-CRISPR/Cas9 nanosystem for sono-immunometabolic cancer therapy. Nature Communications, 13, 7903.

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LPS-Induced Macrophage Analysis in WT and NFIA-Tg Mice

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Normal chow-fed WT or NFIA-Tg mice were treated with either 0.3 mg/kg body weight LPS or saline for 12 h. FACS analysis of iWAT SVF was performed using APC anti-mouse F4/80 antibody (BioLegend, 123116) and FITC anti-mouse/human CD11b antibody (BioLegend, 101205). Flow cytometry and cell sorting were performed using the SH800 cell sorter (SONY).

Hiraike Y., Saito K., Oguchi M., Wada T., Toda G., Tsutsumi S., Bando K., Sagawa J., Nagano G., Ohno H., Kubota N., Kubota T., Aburatani H., Kadowaki T., Waki H., Yanagimoto S, & Yamauchi T. (2023). NFIA in adipocytes reciprocally regulates mitochondrial and inflammatory gene program to improve glucose homeostasis. Proceedings of the National Academy of Sciences of the United States of America, 120(31), e2308750120.

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Multicolor Flow Cytometry Analysis of Immune Cells

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After mice were sacrificed, the desired organs were ground and processed to form a single cell suspension in cell staining buffer. Red blood cell lysis buffer was added to lyse the red blood cell before Fc-receptor blocking. Purified rat anti-mouse CD16/CD32 (BD Pharmingen) was used to block non-specific staining of antibodies. After blocking, FITC anti-mouse/human CD11b antibody (BioLegend), brilliant violet 421 anti-mouse CD19 antibody (BioLegend), and PE anti-mouse CD3 antibody (BioLegend) were used to labeled macrophages, B cell, and T cells, and the pool of cells were analyzed and sorted by FACS (BD FACSAriz II).

Lee Y.W., Mout R., Luther D.C., Liu Y., Castellanos-García L., Burnside A.S., Ray M., Tonga G.Y., Hardie J., Nagaraj H., Das R., Phillips E.L., Tay T., Vachet R.W, & Rotello V.M. (2019). In Vivo Editing of Macrophages through Systemic Delivery of CRISPR-Cas9-Ribonucleoprotein-Nanoparticle Nanoassemblies. Advanced therapeutics, 2(10), 1900041.

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In Vivo Apoptosis and MDSC Analysis

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To investigate the in vivo drug effects, flow cytometry was used to analyze the apoptosis changes and detect myeloid-derived suppressor cells (MDSCs) in the abdomen. On the 48th day after the 1st drug administration, the mice were then sacrificed, ascites or peritoneal washing fluid were collected, and the pellet was separated via centrifuging. An Annexin V/PI Apoptosis Kit was used to analyze apoptosis changes and compare differences in each treatment group. PE anti-mouse CD45 antibody (BioLegend, San Diego, CA, USA, 103,106/200 µg), APC anti-mouse Ly-6G/Ly-6C (Gr-1) antibody (BioLegend, 108,412/100 µg) and FITC anti-mouse/human CD11b antibody (BioLegend, 101,206/500 µg) were used as antibodies to detect MDSCs. Then, samples were subjected to flow cytometry analysis under the guidance of the manufacturer’s protocol.
Ascites, peritoneal washing fluid and tumor tissues from different treatment groups were stained using Caspase 3 Polyclonal antibody and Anti-VEGFA antibody to detect caspase 3 and VEGFA via IHC (methods described in Section 2.2).

Yang B., Yin S., Zhou Z., Huang L, & Xi M. (2023). Inflammation Control and Tumor Growth Inhibition of Ovarian Cancer by Targeting Adhesion Molecules of E-Selectin. Cancers, 15(7), 2136.

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Isolation and Characterization of Peri-Infarct Cells

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The peri-infarct tissues of mice were collected and then temporarily placed on ice. Tissues were dissociated and digested for 1 h at 37 °C by Papain (2 mg/mL, LS003119, Worthington) in RPMI 1640 medium (C11875500BT, Gibico). The mixture was passed through a 70-μm nylon mesh. Dispersed cells were collected by centrifugation with 300 × g for 10 min. The cell pellet was resuspended in 30% Percoll density gradient (17089109, Cytiva) and centrifuged at 900 × g for 25 min. Samples were blocked with FcR Blocking Reagent (130-092-575, Miltenyi Biotec) and resuspended in PBS containing 2% FBS. The different cells were assayed for surface antigens by flow cytometry as previously described^{17 (link)}. Cells were used for fluorescence acquisition and stained with APC anti-Mouse NCAM-1/CD56 Allophycocyanin MAb (FAB7820A-100, R&D), PE anti-mouse ACSA-2 (130-116-244, Miltenyi Biotec), FITC anti-mouse/human CD11b Antibody (101205, BioLegend), PerCp-CyTM5.5 anti-mouse CD45 Antibody (561869, BD Pharmingen), and Brilliant Violet 605™ anti-mouse CD31 (102427, BioLegend). Gating was determined based on the appropriate negative isotype-stained controls. Flow cytometry (BD Biosciences, FACSAria II SORP) was used for fluorescence acquisition, and data were analyzed using FlowJo-V10 software.

Li B., Xi W., Bai Y., Liu X., Zhang Y., Li L., Bian L., Liu C., Tang Y., Shen L., Yang L., Gu X., Xie J., Zhou Z., Wang Y., Yu X., Wang J., Chao J., Han B, & Yao H. (2023). FTO-dependent m6A modification of Plpp3 in circSCMH1-regulated vascular repair and functional recovery following stroke. Nature Communications, 14, 489.

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Isolation and Characterization of Brain Cell Types

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Isolated brain cells were prepared from C57BL/6J mice. Tissue was digested by papain at 37 °C for 1 h (2 mg/mL, LS003119, Worthington, USA) in DMEM medium. Dispersed cells were filtrated with a nylon mesh (70 μm). The cells were resuspended in Percoll density gradient (30%, 17-0891-09, GE Healthcare, USA) and centrifuged (900×g) at 25 °C for 25 min. Next, the cells in the bottom were collected. After washing in PBS containing 2% FBS, the cells were blocked with FcR Blocking Reagent (130-092-575, Miltenyi Biotec). Astrocytes, microglial cells, neurons, and endothelial cells were marked by flow cytometry42 (link), 43 (link), 44 (link), 45 (link). Cells were stained with PE anti-mouse ACSA-2 (130-116-244, Miltenyi Biotec, Germany), FITC anti-mouse/human CD11b antibody (101205, BioLegend, USA), PerCp-cy5.5 anti-mouse CD45 antibody (561869, BD Pharmingen, USA), APC anti-mouse NCAM-1/CD56 allophycocyanin MAb (FAB7820A-100, R&D, USA), and Brilliant Violet 605™ anti-mouse CD31 (102427, BioLegend, USA). After staining, the samples were sorted by FACSAria II SORP (BD Biosciences, USA), and the data were analyzed using FlowJo_V10 (FlowJo). Samples were gated for ACSA-2⁺ (astrocytes), ACSA-2^–CD11b⁺CD45^dim (microglial cells), NCAM-1/CD56⁺ (neurons), and CD31⁺ (endothelial cells). The RNeasy®-Micro Kit (74004, QIAGEN, Germany) was used for RNA extraction.

Bai Y., Chang D., Ren H., Ju M., Wang Y., Chen B., Li H., Liu X., Li D., Huo X., Guo X., Tong M., Tan Y., Yao H, & Han B. (2024). Engagement of N6-methyladenisine methylation of Gng4 mRNA in astrocyte dysfunction regulated by CircHECW2. Acta Pharmaceutica Sinica. B, 14(4), 1644-1660.

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Multicolor Flow Cytometry Analysis of Immune Cells

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The cells were analysed by cytoFLEX flow cytometer (Beckman Coulter, USA), using the FlowJo software. The antibodies used in present study were FITC anti‐mouse/human CD11B Antibody (Biolegend, Cat. No. 101205, USA), APC anti‐mouse F4/80 Antibody (Biolegend, Cat. No. 123116, USA), APC anti‐mouse CD206 (MMR) Antibody (Biolegend, Cat. No. 141707, USA) and PE anti‐mouse Ly6G Monoclonal Antibody (Elabscience, Cat. No. E‐AB‐F1108D, China).

Li K., Xu W., Lu K., Wen Y., Xin T., Shen Y., Lv X., Hu S., Jin R, & Wu X. (2020). CSF‐1R inhibition disrupts the dialog between leukaemia cells and macrophages and delays leukaemia progression. Journal of Cellular and Molecular Medicine, 24(22), 13115-13128.

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Quantifying M2-like Tumor-Associated Macrophages

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To measure the percentage of M2-like TAMs, excised tumours from various tumour models were prepared for single-cell suspension after in vivo fluorescence imaging by the IVIS Spectrum imaging system. The samples were stained with PerCP/Cy5.5 anti-mouse CD45 antibody (103132, Biolegend, clone number: 30-F11, Dilution 1:100), FITC anti-mouse/human CD11b antibody (101205, Biolegend, clone number: M1/70, Dilution 1:200), PE/Cy7 anti-mouse F4/80 antibody (123114, Biolegend, clone number: BM8, Dilution 1:100), and PE anti-mouse CD206 antibody (141706, Biolegend, clone number: C068C2, Dilution 1:40) according to the manufacturer’s instructions. The proportions of CD11b⁺F4/80⁺CD206⁺ macrophages were obtained by flow cytometry, and the correlation analysis between M2-like macrophage content and the ratiometric signal was measured by GraphPad Prism 8 software.

Tang M., Chen B., Xia H., Pan M., Zhao R., Zhou J., Yin Q., Wan F., Yan Y., Fu C., Zhong L., Zhang Q, & Wang Y. (2023). pH-gated nanoparticles selectively regulate lysosomal function of tumour-associated macrophages for cancer immunotherapy. Nature Communications, 14, 5888.

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