Anti cd11c antibody

Manufactured by BioLegend

Sourced in United States

The Anti-CD11c antibody is a laboratory reagent used to identify and study cells expressing the CD11c antigen. CD11c is a marker for dendritic cells and macrophages, and this antibody can be used in flow cytometry and other immunoassays to detect and analyze these cell types.

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6 protocols using anti cd11c antibody

Characterization of DC Subsets Uptake of CpG ODNs

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Mouse bone marrow-derived DCs (1 × 10⁷ cells/mL in a 96-well flat-bottom culture plate) were treated with Alexa 488-labeled K-type CpG ODNs (1.25 µg CpG ODNs/mL) or CA nanoparticles containing Alexa 488-labeled K-type CpG ODNs (CA-Alexa 488-CpG; 1.25 µg CpG ODNs/mL) for 10, 30, 90, 120, or 180 min. Cells were stained with 0.4 w/v% trypan blue solution (Wako, Osaka, Japan) to quench any Alexa 488-labeled K-type CpG ODNs bound to the cell surface, and then the cells were analyzed by means of flow cytometry (NovoCyte Flow Cytometer, ACEA Bioscience, San Diego, CA, USA). To separate various subsets of DCs from the collected cells, we incubated the cells with anti-mouse CD16/CD32 antibody (TONBO biosciences, San Diego, CA, USA), anti-mouse B220 antibody (BioLegend), anti-CD11c antibody (BioLegend), and anti-CD11b antibody (BioLegend) in the absence of trypan blue. In this way, the DCs were separated into the following subsets: B220⁺ CD11c⁺ plasmacytoid DCs, CD11b⁺ CD11c⁺ conventional DCs, and CD11b⁻ CD11c⁺ conventional DCs.

Takahashi H., Misato K., Aoshi T., Yamamoto Y., Kubota Y., Wu X., Kuroda E., Ishii K.J., Yamamoto H, & Yoshioka Y. (2018). Carbonate Apatite Nanoparticles Act as Potent Vaccine Adjuvant Delivery Vehicles by Enhancing Cytokine Production Induced by Encapsulated Cytosine-Phosphate-Guanine Oligodeoxynucleotides. Frontiers in Immunology, 9, 783.

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BMDC Activation by Coculture with Treated CT26 Cells

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BMDCs were generated from the bone marrow of 10 week old BALB/c mice. 7 × 10⁵ BMDCs were seeded in 12‐well plates and cocultured with HP, FHP‐1, and FHP‐2 pretreated CT26 cells for 24 h. After incubation, the cells were collected and blocked with antimouse CD16/32 antibody (Biolegend, CA, USA) for 10 min. Subsequently, the cells were stained with anti‐CD11c antibody, anti‐CD40 antibody, anti‐CD80 antibody, and anti‐CD86 antibody (Biolegend) for 30 min. The stained cells were analyzed by flow cytometry (FACS Fortessa, BD Biosciences).

Jeong S.D., Jung B., Ahn H.M., Lee D., Ha J., Noh I., Yun C, & Kim Y. (2021). Immunogenic Cell Death Inducing Fluorinated Mitochondria‐Disrupting Helical Polypeptide Synergizes with PD‐L1 Immune Checkpoint Blockade. Advanced Science, 8(7), 2001308.

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Flow Cytometry Analysis of CpG-induced DC Subsets

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C57BL/6J mice were treated with CpG ODN (10 μg/mouse) or each LNP-CpG (10 μg CpG ODN/mouse) subcutaneously at the base of the tail. Twenty-four hours after administration, the lymph nodes draining the site of administration were collected after euthanasia. To prepare single-cell suspensions, the draining lymph nodes were incubated with 200 μg/mL Liberase TL (Roche Diagnostics GmbH, Mannheim, Germany) and 10 U/mL DNase I (Roche Diagnostics GmbH) for 60 min at 37°C. Prepared cells were incubated with anti-mouse CD3ε antibody (BioLegend), anti-mouse CD19 antibody (BioLegend), anti-mouse CD16/CD32 antibody, anti-CD11c antibody, anti-PDCA-1 antibody (BioLegend), anti-CD80 antibody, or anti-CD86 antibody for flow cytometry. In this way, the DCs were separated into two subsets, namely CD3ε⁻ CD19⁻ PDCA-1⁺ CD11c⁺ pDCs and CD3ε⁻ CD19⁻ PDCA-1⁻ CD11c⁺ conventional DCs (cDCs). The cells were then analyzed by means of flow cytometry.

Shirai S., Shibuya M., Kawai A., Tamiya S., Munakata L., Omata D., Suzuki R., Aoshi T, & Yoshioka Y. (2020). Lipid Nanoparticles Potentiate CpG-Oligodeoxynucleotide-Based Vaccine for Influenza Virus. Frontiers in Immunology, 10, 3018.

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Characterization of Immune Landscape in 4T1 Tumor Model

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The livers and bone marrow cells were harvested from sham and 4T1 cancer-bearing animals 14 days after transplantation. Red blood cells (RBCs) were lysed with RBC lysis buffer (BD Bioscience). The obtained cells were then stained with the following antibodies (BioLegend, CA, USA) for 30 min at 4 °C: anti-CD45 antibody (Clone: 30-F11), anti-CD19 antibody (Clone: 6D5), anti-TCRβ antibody (Clone: H57-597), anti-CD11b antibody (Clone: M1/70), anti-Ly6G antibody (Clone: 1A8), anti-FCεR1a antibody (Clone: MAR-1), anti-CD117 antibody (Clone: 2B8), anti-CD123 (Clone: 5B11), anti-CD49b antibody (Clone: DX5), anti-CD3 antibody (Clone: 17A2), anti-CD11c antibody (Clone: N418), anti-TER-119 antibody, or anti-CD34 antibody (Clone: HM34). Non-viable cells were stained with Propidium Iodide Solution (PI) and gated out. Data were acquired using FACS Aria (BD Bioscience) and analyzed using FlowJo software (v10.8.1, BD Biosciences). Gating strategy is shown in Supplementary Fig. 14.

Vandenbon A., Mizuno R., Konishi R., Onishi M., Masuda K., Kobayashi Y., Kawamoto H., Suzuki A., He C., Nakamura Y., Kawaguchi K., Toi M., Shimizu M., Tanaka Y., Suzuki Y, & Kawaoka S. (2023). Murine breast cancers disorganize the liver transcriptome in a zonated manner. Communications Biology, 6, 97.

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Generation and Characterization of Bone Marrow-Derived Dendritic Cells

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To generate bone-marrow-derived DCs, we isolated bone marrow cells from the femurs of C57BL/6J mice and cultured the cells at 37°C for 7 days with 100 ng/mL human Fms-related tyrosine kinase 3 ligand (PeproTech, Rocky Hill, NJ, USA). Cells were seeded at a density of 1 × 10⁵ cells/well in a 96-well flat-bottomed culture plate (Nunc, Roskilde, Denmark) and were cultured in complete RPMI medium (RPMI 1640 supplemented with 10 vol. % fetal calf serum, penicillin, and streptomycin). These cells were stimulated with CpG ODN or with each LNP-CpG for 24 h. Supernatants were subjected to ELISA to determine the levels of IFN-α (InvivoGen) and interleukin (IL)-12 p40 (BioLegend, San Diego, CA, USA), in accordance with the manufacturers' instructions. To check the levels of co-stimulatory molecules on DCs, we incubated the cells with anti-mouse CD16/CD32 antibody (BioLegend), anti-CD11c antibody (BioLegend), anti-CD11b antibody (BioLegend), anti-CD80 antibody (BioLegend), or anti-CD86 antibody (BioLegend). Then the cells were analyzed by means of flow cytometry (NovoCyte Flow Cytometer, ACEA Biosciences, San Diego, CA, USA).

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Integrin-Mediated Monocyte Adhesion

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The role of CD11b/CD18 and CD11c/CD18 in the adhesion to fibrinogen was analysed by comparing the adhesive properties of monocytes, MDMs and MDDCs treated with either anti-CD11b antibody (monoclonal mIgG1 clone TMG6-5, provided by István Andó at BRC Szeged, Hungary) or anti-CD11c antibody (monoclonal mIgG1 clone 3.9, Biolegend). Both antibodies are specific for the ligand binding domain of the integrins and were used in sterile, azide-free form at saturating concentration previously titrated by flow cytometry. Cells were incubated with the receptor-specific antibodies for 30min at 4°C and used in adhesion studies without washing. Since unoccupied integrins are known to recycle to the cell surface, and would decrease the efficiency of blocking, unbound antibodies were not washed away. Cells were incubated in the presence of FcR blocking reagent (Miltenyi Biotech), and the effect of receptor specific antibodies was compared to untreated samples that were incubated only with FcR blocking reagent.

Sándor N., Lukácsi S., Ungai-Salánki R., Orgován N., Szabó B., Horváth R., Erdei A, & Bajtay Z. (2016). CD11c/CD18 Dominates Adhesion of Human Monocytes, Macrophages and Dendritic Cells over CD11b/CD18. PLoS ONE, 11(9), e0163120.

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