Anti cd11c

Manufactured by Miltenyi Biotec

Sourced in Germany

Anti-CD11c is a laboratory equipment product from Miltenyi Biotec. It is used for the detection and isolation of cells expressing the CD11c antigen, which is a surface marker found on dendritic cells and other myeloid cells.

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Anti-CD11c microbeads (62 citations) Anti-CD11c magnetic beads (18 citations) Anti-mouse CD11c microbeads (11 citations) Anti-CD11c MACS beads (8 citations) Anti-CD11c beads (7 citations) Anti-CD11c magnetic microbeads (6 citations) Anti-CD11c-coated magnetic beads (4 citations) Anti-CD11c immunomagnetic beads (4 citations) Anti-CD11c MACS microbeads (4 citations) Bead-conjugated anti-CD11c (4 citations)

13 protocols using anti cd11c

Comprehensive C2C12 Myoblast Profiling

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Cell cycle/Edu experiments were performed by incubating growth phase C2C12 myoblast cultures with 10uM 5-ethynyl-2′-deoxyuridine (EdU) for 2 hours, fixing, counterstaining DNA with DAPI, and identifying Edu+ cells using a Click-IT Edu Flow Cytometry Kit (Thermo Fisher). Mitochondria were labeled in live cells using MitoTracker Green FM (Thermo Fisher) and analyzed by flow cytometry. Immune cell profiling was performed as follows: hindlimb muscles were enzymatically digested and filtered according to standard protocols (Bernet et al, 2014 ), cells were labeled with fluorescently conjugated primary antibodies according to manufacturer’s instructions, and flow cytometry performed on a MACSquant 10 analyzer. The following antibodies (Miltenyi Biotec) were used in this study: anti-CD45, anti-CD3, anti-CD49b, anti-CD11c, anti-MHC class II, anti-F4/80, anti-CD11c, and anti-GR1. The following is the gating strategy used to identify individual immune cell subtypes: NK: CD45+, CD3e−, CD49b+; Dendritic cell: CD45+, CD11c+, MHC class II+; M1 macrophage: CD45+, F4/80+, CD11b+, CD11c+; M2 macrophage: CD45+, F4/80+, CD11b+, CD11c−; Neutrophil: CD45+, CD11b+, GR1+; Pan T cells: CD45+, CD3e+; Cytotoxic T cells: CD45+, CD3e+, CD8+; Helper T cells: CD45+, CD3e+, CD4+; Regulatory T cells: CD45+, CD3e+, CD4+, CD25+. Samples were analyzed using either MACSquant software or FlowJo v10.

Hogan K.A., Cho D.S., Arneson P.C., Samani A., Palines P., Yang Y, & Doles J.D. (2017). Tumor-derived cytokines impair myogenesis and alter the skeletal muscle immune microenvironment. Cytokine, 107, 9-17.

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Isolation of Lung Antigen-Presenting Cells

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Mouse lung, draining lymph node or spleen single-cell suspensions were prepared by mincing through a 40-μm Falcon cell strainer, followed by RBC lysis (ACK lysis buffer) for 3 minutes. To isolate lung APCs, RBC-free whole lung cells were labeled with paramagnetic bead– conjugated anti-CD11c (Miltenyi Biotec) and isolated using autoMACS.

Hong M.J., Gu B.H., Madison M., Landers C., Tung H.Y., Kim M., Yuan X., You R., MacHado A.A., Gilbert B.E., Soroosh P., Elloso M., Song L., Chen M., Corry D.B., Diehl G, & Kheradmand F. (2017). Protective Role of γδ T Cells in Cigarette Smoke and Influenza Infection. Mucosal immunology, 11(3), 894-908.

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Isolation of Lung Antigen-Presenting Cells

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Adoptive Transfer of Alveolar Macrophages

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AMs were enriched by positive magnetic selection using anti-CD11c (Miltenyi Biotech). The positively selected fraction was collected after passing through the LS column (Miltenyi Biotech) on an MACS magnetic separator (Miltenyi Biotech) and plated with DMEM with 10% fetal calf serum, L-glutamine, penicillin, and streptomycin. AMs were kept for 2 h to adhere and washed with warm media. The purity of the cells was confirmed by flow cytometry. From this, 2 × 10⁶ macrophages were transferred per recipient. The adoptive transfer was performed in 50 μl of sterile PBS by i.t. delivery into mice anesthetized with ketamine/xylazine. Before transfer, host AMs were not depleted to avoid bystander inflammatory response (Aegerter et al., 2020 (link)).

Chakraborty S., Singh A., Wang L., Wang X., Sanborn M.A., Ye Z., Maienschein-Cline M., Mukhopadhyay A., Ganesh B.B., Malik A.B, & Rehman J. (2023). Trained immunity of alveolar macrophages enhances injury resolution via KLF4-MERTK-mediated efferocytosis. The Journal of Experimental Medicine, 220(11), e20221388.

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Purification of Splenic B Cells and CD4+ T Cells

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Splenic B cells were purified magnetically by positive selection with anti-CD19 microbeads after negative selection by a mixture of anti-CD90.2, anti-CD11c and anti-Ter119 microbeads (Miltenyi Biotec, Auburn, CA) (greater than 99.5% pure and 90% viable). CD4⁺ T cells were isolated by a CD4⁺ T cell isolation kit (Miltenyi Biotec) (more than 94.7% pure and 95% viable). In some experiments, unfractionated CD4⁺ T cells were further fractionated into CD25⁺ and CD25⁻ T cells by PE-conjugated anti-CD25 antibody with anti-PE microbeads. Red blood cell lysed-unfractionated splenocytes from (Il10^+/+) Rag2^−/− and DKO mice were used for WT and Il10^−/− antigen presenting cells (APC), respectively (more than 88.4% CD11b⁺).

Mishima Y., Liu B., Hansen J.J, & Sartor R.B. (2015). Resident Bacteria-Stimulated Interleukin-10-Secreting B Cells Ameliorate T-Cell-Mediated Colitis by Inducing T-Regulatory-1 Cells That Require Interleukin-27 Signaling. Cellular and Molecular Gastroenterology and Hepatology, 1(3), 295-310.

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Purification of Splenic B and T Cells

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Splenic B cells were purified magnetically by positive selection with anti-CD19 microbeads after negative selection by a mixture of anti-CD90.2, anti-CD11c, and anti-Ter119 microbeads (Miltenyi Biotec, Auburn, CA) (greater than 99.5% pure and 90% viable). The CD4⁺ T cells were isolated by a CD4⁺ T-cell isolation kit (Miltenyi Biotec) (more than 94.7% pure and 95% viable). In some experiments, unfractionated CD4⁺ T cells were further fractionated into CD25⁺ and CD25⁻ T cells by PE-conjugated anti-CD25 antibody with anti-PE microbeads. Red blood cell lysed-unfractionated splenocytes from (Il10^+/+)Rag2^−/− and DKO mice were used for WT and Il10^−/− antigen-presenting cells (APC), respectively (more than 88.4% CD11b⁺).

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Isolation of Murine B Cell Subsets

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Human PBMC purified B cells were isolated by Ficoll-Paque, followed by anti-CD19 magnetic beads (Miltenyi Biotec). Splenic naive B cells were purified using a B cell isolation kit by negative selection (Miltenyi Biotec). Splenic Pre-GC and GC B cells were first enriched by streptavidin magnetic beads conjugated with a mixture of biotinylated anti-CD11b, anti-CD11c, anti-IgD, anti-CD138, anti-CD3, and anti-Ter119 (Miltenyi Biotec), followed by cell sorting using a BD FACSAria III Cell Sorter. Splenic Pre-GC cells defined as B220⁺ IgD⁻ CD38⁺ GL7⁺ cells were isolated from PyNL-infected mice on day 9 after infection. Splenic mature GC B cells defined as B220⁺ IgD⁻ CD38⁻ GL7⁺ cells were purified from mice recovered from PyNL infection after day 28 infection.

Lee M.S., Inoue T., Ise W., Matsuo-Dapaah J., Wing J.B., Temizoz B., Kobiyama K., Hayashi T., Patil A., Sakaguchi S., Simon A.K., Bezbradica J.S., Nagatoishi S., Tsumoto K., Inoue J.I., Akira S., Kurosaki T., Ishii K.J, & Coban C. (2021). B cell–intrinsic TBK1 is essential for germinal center formation during infection and vaccination in mice. The Journal of Experimental Medicine, 219(2), e20211336.

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Isolation of Retinal Ganglion Cells

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Retinal ganglion cells were isolated using the Miltenyi Adult Brain Dissociation Kit (Miltenyi Biotec, 130-107-677) and MACS cell separation system. Briefly, whole neurosensory retinas were harvested, and retinal tissues were dissociated in manufacturer-provided enzyme mixtures using the gentle MACS dissociator pre-set program: 37C_ABDK_01 (Miltenyi Biotec). Dissociated retinal cell suspensions were filtered using a 70 μm filter (Miltenyi Biotec, 130-098-462) and resuspended in debris removal solution. Retinal cells were resuspended and incubated in red blood cell removal solution for 10 minutes at 4 °C. The resulting cell suspension was then subjected to a selection protocol using MACS magnetic separation LS columns (Miltenyi Biotec, 130-042-401) and the following antigen-coated magnetic microbeads: (1) anti-CD11c (Miltenyi Biotec, 130-108-338), (2) anti-ACSA2 (Miltenyi Biotec, 130-097-678), (3) anti-CD45 (Miltenyi Biotec, 130-052-301), (4) anti-CD15 (Miltenyi Biotec, 130-094-530), and a final positive selection step (5) anti-Thy1 (Miltenyi Biotec, 130-121-278), using the manufacturer’s protocol to isolate the final target cell population: CD11c− ACSA2− CD45− CD15− Thy1+. RGC enrichment in the final cell fraction was confirmed using qPCR.

Zhang K.R., Baumann B., Song Y., Sterling J., Erler E.A., Guttha S., Kozmik Z, & Dunaief J.L. (2022). Conditional knockout of hephaestin in the neural retina disrupts retinal iron homeostasis. Experimental eye research, 218, 109028.

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Ex vivo Phagocytosis Assay of CD4+ T Cells

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Splenocytes isolated on day 21 of LCMV-Cl13 infection were pooled from multiple mice and then B cell and T cell-depleted using anti-CD19, anti-Thy1.2, and anti-CD4 magnetic beads (Miltenyi Biotec) and allowed to recover for 1hr at 37^oC in complete media. Splenocytes were pooled from multiple mice for each experiment to obtain enough cells for the ex vivo phagocytosis assay and experiments were repeated multiple times. B cell/T cell-depleted splenocytes or peritoneal macrophages were mixed with negatively selected, naïve CD45.1⁺ CD4 T cell targets at a 100:1 splenocyte:T cell or 50:1 peritoneal macrophage:T cell ratio. 30μg/ml of isotype or anti-CD4 (clone GK1.5) Ab were added to the culture and the number of target cells was assessed by flow cytometry two days later. In some assays, DC or macrophages were removed using anti-CD11c or F4/80-PE staining followed by anti-PE magnetic beads, respectively (Miltenyi Biotec).

Yamada D.H., Elsaesser H., Lux A., Timmerman J.M., Morrison S.L., de la Torre J.C., Nimmerjahn F, & Brooks D.G. (2015). Suppression of Fcγ-Receptor-Mediated Antibody Effector Function during Persistent Viral Infection. Immunity, 42(2), 379-390.

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Isolation and Culture of Mouse ILC1s

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ILC1s were isolated from the spleens of C57BL/6 mice by combining magnetic beads and flow sorting. Mouse spleen cells were used to generated single-cell splenocyte suspensions[24 ]. The obtained cells were incubated with biotin-conjugated antibodies (anti-CD3ε, anti-CD45R, anti-Gr-1, anti-CD11c, anti-CD11b, anti-Ter119, anti-TCR-αβ, and anti-FCεRI; Miltenyi, Belgish, Germany) to enrich lineage-negative cells following the Miltenyi magnetic bead cell isolation protocol. Enriched lineage-negative single cell suspensions were stained with an anti-mouse lineage cocktail (BioLegend, 145-2C11, RB6-8C5, RA3-6B2, Ter-119, and M1/70), anti-mouse CD127 (BioLegend, A7R34), anti-mouse NK1.1 (BioLegend, PK136), and anti-mouse NKp46 (BioLegend, 29A1.4) for flow cytometry sorting (BD FACS Melody) of lineage⁻ CD127⁺ NK1.1⁺ NKp46⁺ cells (which were considered ILC1s); these cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 0.5 ng/ml IL-7 (Gibco, PMC0071).

Zhang Y., Ma S., Li T., Tian Y., Zhou H., Wang H, & Huang L. (2023). ILC1-derived IFN-γ regulates macrophage activation in colon cancer. Biology Direct, 18, 56.

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