Anti cd11c

Manufactured by Thermo Fisher Scientific

Sourced in United States

Anti-CD11c is a laboratory reagent used for the detection and identification of CD11c-expressing cells, such as dendritic cells and some monocytes, via flow cytometry or other immunoassay techniques. It provides a tool for researchers to study the phenotype and function of these cell populations in various biological samples.

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67 protocols using anti cd11c

Flow Cytometry Analysis of BMNC

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BMNC isolated as described²⁸ were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD11b, anti-CD11c, anti-CD45, anti-F4/80, anti-Gr-1 or appropriate isotype control mAb (all from eBiosciences). CFSE (0.7 mg in 0.3 ml PBS-DMSO 7%; Santa Cruz Biotechnologies) or vehicle were injected i.v. on day 5 post-infection as reported^{3 (link)}. BMNC were then collected and stained with anti-CD11b, anti-CD11c or isotype control mAb (eBiosciences) and examined for expression of CFSE. Splenocytes were incubated with or without anti-CD3 mAb and Brefeldin A (10 μg/ml; eBiosciences). Cells were stained with anti-CD3, anti-CD4 and anti-CD8 (eBiosciences), permeabilized, stained with anti-IFN-γ or isotype control mAb (eBiosciences) and analyzed in an LSR II flow cytometer (BD Biosciences).

Corcino Y.L., Portillo J.A, & Subauste C.S. (2019). Epidermal growth factor receptor promotes cerebral and retinal invasion by Toxoplasma gondii. Scientific Reports, 9, 669.

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Comprehensive Immune Cell Profiling in Mice

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Immune cells of 3-month-old male mice were isolated from spleen, kidney, bone marrow and thymus by flushing and pelleting (bone marrow) or by mechanical dissociation followed by centrifugation through a percoll gradient (kidney, spleen and thymus). Erythrocytes were lysed with ammonium-chloride-potassium lysing buffer, and the resulting leukocytes were counted on a haemocytometer. Cells were stained for flow cytometry analysis with anti-CD45 (Becton Dickinson, ref: 25-0451-82, dilution: 1/1,000), anti-B220 (eBioscience, ref: 45-0452-80, dilution: 1/1,000), anti-CD11b (eBioscience, ref: 12-0112-82, dilution: 1/1,000), anti-CD11c (eBioscience, ref: 11-0114-85, dilution: 1/1,000), anti-NKp46 (eBioscience, ref: 48-3351-82, dilution: 1/1,000), anti-CD8 (eBioscience, ref: 17-0081-81, dilution: 1/1,000), anti-TCRβ (Becton Dickinson, ref: 563221, dilution: 1/1,000) and Fixable Viability Dye (eBioscience, ref: 65-0865-14). Samples were acquired on CyAnADP 9 flow cytometer (Beckman Coulter) using Summit acquisition software (V). Data were analysed using Kaluza flow analysis software V (Beckman Coulter). An example of this analysis is shown in Supplementary Fig. 12.

Messaoudi S., He Y., Gutsol A., Wight A., Hébert R.L., Vilmundarson R.O., Makrigiannis A.P., Chalmers J., Hamet P., Tremblay J., McPherson R., Stewart A.F., Touyz R.M, & Nemer M. (2015). Endothelial Gata5 transcription factor regulates blood pressure. Nature Communications, 6, 8835.

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Flow Cytometric Immunophenotyping of NPCs

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FACSCalibur flow cytometer (BD Biosciences) was employed for flow cytometry. Firstly, NPCs’ Fcγ III/II receptor were blocked by anti-CD16/CD32 antibodies. Then, incubated with anti-CD11c, anti-CD3, anti-CD19 and anti-F4/80 antibodies (eBiosciences), respectively. The data was analyzed with CELLQuest software (BD Biosciences).

Fang J.Z., Li C., Liu X.Y., Hu T.T., Fan Z.S, & Han Z.G. (2015). Hepatocyte-Specific Arid1a Deficiency Initiates Mouse Steatohepatitis and Hepatocellular Carcinoma. PLoS ONE, 10(11), e0143042.

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Flow Cytometry Analysis of Myeloid Cells

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LNs and spleen tissues were prepared using mechanical dissociation of minced tissue to obtain single cell suspensions. The following antibodies were used for flow cytometry: anti-Ly6G/Ly6C (Cat #58-5931-82), anti-Cd11b (Cat #12-0112-82), anti-CD86 (Cat #11-0862-82), anti-F4/80 (Cat #17-4801-82), anti-MHCII (Cat #13-5321-82), and anti-CD11c (Cat #11-0116-42) all from eBioscience, San Diego, CA) and anti-CD206 (clone MR5D3; BioRad). After being stained with a standard protocol, all events were analyzed with FlowJo software (FlowJo, LLC, Ashland, OR) and frequencies among live cells were obtained.

Cheng R., Billet S., Liu C., Haldar S., Choudhury D., Tripathi M., Hav M., Merchant A., Hu T., Huang H., Zhou H, & Bhowmick N.A. (2019). Periodontal inflammation recruits distant metastatic breast cancer cells by increasing myeloid-derived suppressor cells. Oncogene, 39(7), 1543-1556.

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Flow Cytometry Analysis of Murine Immune Cells

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Spleens were homogenized by manual disruption and red cells were lysed as described above. The cells were resuspended at 4°C in FACS buffer (PBS, 5% FBS and 0.1% sodium azide). Antibodies were incubated with cells for 30 min at 4°C at the indicated dilution and washed with FACS buffer before resuspension in fresh FACS buffer. Samples were analyzed on a Cyan flow cytometer (Dako) or an LSR-II flow cytometer (BD). The antibodies used were as follows: anti-Ly6G (48–5931-82; eBioscience; dilution 1/200), anti-CD11c (17–0114-81; eBioscience; dilution 1/200), anti-CD19 (48–0193-82; eBioscience; 1/200), anti CD11b (17–0112-82 or 45–0112-80; eBioscience), anti-CD8a (11–0081-82; eBioscience; 1/400 dilution), anti-CD4 (558107; BD), anti-F4/80 (12–4801-82; eBioscience; 1/200), anti-CD115 (12–1152-82; eBioscience; 1/250), anti CD45.1 (25–0453-82; eBioscience; 1/200), anti-CD45.2 (17–0454-81; eBioscience; 1/200), and anti-Ly6C (53–5932; eBioscience; 1/200).

Thomas D.C., Clare S., Sowerby J.M., Pardo M., Juss J.K., Goulding D.A., van der Weyden L., Storisteanu D., Prakash A., Espéli M., Flint S., Lee J.C., Hoenderdos K., Kane L., Harcourt K., Mukhopadhyay S., Umrania Y., Antrobus R., Nathan J.A., Adams D.J., Bateman A., Choudhary J.S., Lyons P.A., Condliffe A.M., Chilvers E.R., Dougan G, & Smith K.G. (2017). Eros is a novel transmembrane protein that controls the phagocyte respiratory burst and is essential for innate immunity. The Journal of Experimental Medicine, 214(4), 1111-1128.

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Phenotyping Regulatory T Cells and T Follicular Helper Cells

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For CD4⁺CD25⁺Foxp3⁺ T cells, splenocytes were isolated using standard methods. Nuclear stain for transcription factor FoxP3 was performed with the BD Biosciences (San Jose, CA) kit. Briefly, splenocytes (1×10⁶ per mouse) were stained with fluorescent conjugated antibodies, including anti-mouse CD4-pacific blue, anti-mouse CD25-PE and anti-mouse FoxP3-Alexa Fluor 647 according to staining protocol. For TfH cell staining, anti-CXCR5-BV421, anti-CD4-BV605, anti-PD-1-APC, anti-B220-FITC, and anti-Bcl6-PE were purchased from BD Bioscience (San Jose, CA). Fixable viability dye was purchased from eBioscience (San Diego, CA). For dendritic cell characterization, antiCD11b, anti-CD11c, and anti-CD64 were purchased from eBioscience (San Diego, CA). FACS analysis using LSR-II instrumentation from BD Biosciences (San Jose, CA) and the FCS Express software (DeNovo Software).

Herzog R.W., Cooper M., Perrin G.Q., Biswas M M., Martino A.T., Morel L., Terhorst C, & Hoffman B.E. (2017). Regulatory T cells and TLR9 Activation Shape Antibody Formation to a Secreted Transgene Product in AAV Muscle Gene Transfer. Cellular immunology.

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Multiparametric Flow Cytometry Analysis of Immune Cells

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Cells were incubated with antibodies 4 °C 20 min. Cells were examined by Aria II Flow Cytometer (BD Bioscience, USA). Cells were gated as follows: DCs (F4/80⁻ CD11c⁺ IA/IE⁺), macrophages (CD11b⁺ F4/80⁺) and M1 macrophages (CD11b⁺ F4/80⁺ CD206⁻ MHC II^high), M2 macrophages (CD11b⁺ F4/80⁺ CD206⁺ MHC II^low). Intracellular cytokine staining: 2 µL mL⁻¹ Cell Activation Cocktail (with Brefeldin A) (Cat: 423,303, Biolegend, USA) was used to incubate cells at 37 °C in a CO₂ incubator for 6 h. Then, the Fixation/Permeabilization Solution Kit (Cat: 554,714, BD Biosciences, USA) was used to stimulus cells. After cell fixation and permeabilization (fixation/permeabilization solution, 100 uL/10⁶ cells, 4 °C, 30 min), the BD Perm/Wash™ Buffer is used to wash the cells and to dilute the IFN-γ antibody for staining (4 °C, 30 min). Antibodies: The following were purchased from BioLegend: anti-IA/IE (1:3200, Clone: M5/114.15.2, Cat: 107,630), anti-CD206 (1:200, Clone: C068C2, Cat: 141,705), anti-Ki67 (1:200, Clone: 16A8, Cat: 652,403), anti-IFN-γ (1:100, Clone: XMG1.2, Cat: 505,805). The following were purchased from eBioscience: anti-CD11b (1:200, Clone: M1/70, Cat: 47–0112-82), anti-CD11c (1:200, Clone: N418, Cat: 45–0114-82), anti-F4/80 (1:200, Clone: BM8, Cat: 17–4801-82).

Zhao Q., Shi M., Yin C., Zhao Z., Zhang J., Wang J., Shen K., Zhang L., Tang H., Xiao Y, & Zhang Y. (2020). Dual-Wavelength Photosensitive Nano-in-Micro Scaffold Regulates Innate and Adaptive Immune Responses for Osteogenesis. Nano-Micro Letters, 13, 28.

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Whole Blood and Spleen Cell Isolation and Staining

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Peripheral blood (50 μL) was collected and placed in 4 ml of mouse RBC lysis buffer (eBioscience). Splenic single-cell suspensions were prepared by mechanical dissociation of the spleen, followed by filtration, and lysis of the RBCs. Cells were stained with anti-mouse CD4, CD8α, CD19, CD3e, NK1.1, λσ-T, FoxP3, and granzyme B antibodies (eBiosciences). For granzyme B measurements, T cell activation was performed before Ab staining. Splenic single-cell suspensions were prepared and cultured in RPMI 1640 (containing 10% FBS) plus IL-2. T cells were activated for 7 days using a Dynabead Mouse T Activator CD3/CD28 kit. Antibodies used for flow cytometry in this study (anti-CD11c, anti-CD11b, anti-CD19, anti-NK1.1, anti-CD3, and anti-MHCII) were from eBioscience, and anti-TNF-α was from Becton-Dickinson. Single-cell suspensions were prepared with a Neural Tissue Dissociation Kit from Miltenyi Biotec. For CD11b and TNF-α analysis, cells were treated with Cell Stimulation Cocktail and a protein transport inhibitor (eBioscience) overnight, stained for surface CD11b, and fixed and permeabilized for TNF-α detection (using flow cytometry). TMEM 119 was used to stain microglia via ex vivo flow cytometry and by immunohistochemistry (34 (link)).

Rao G., Latha K., Ott M., Sabbagh A., Marisetty A., Ling X., Zamler D., Doucette T.A., Yang Y., Kong L.Y., Wei J., Fuller G.N., Benavides F., Sonabend A.M., Long J., Li S., Curran M, & Heimberger A.B. (2020). Anti-PD-1 induces M1 polarization in the glioma microenvironment and exerts therapeutic efficacy in the absence of CD8 cytotoxic T cells. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(17), 4699-4712.

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Immunomodulatory Protocols for Cancer Research

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Antibodies used in this study were anti-DNMT1 (Abcam), anti-5mC (Cell Signaling), anti-GAPDH, anti-ERK, anti-phospho-ERK (Cell signaling), anti-SMA (Sigma), anti-CD8 (Biolegend, BioXcell, or eBiosciences), anti-CD11c, anti-CD45, anti-F480, anti-MECA-79, anti-Vcam1, and anti-CD4 (all from BD Biosciences), anti-granzyme B (Cell Signaling), anti-mouse IgG (BioXcell), anti-PD-1 (CD279, BioXcell), and anti-PD-L1 (Genentech, MTA program). MGECs were isolated and cultured as described previously by our lab^{60 ,61 (link)}. Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza and cultured in EBM-2 supplemented with the EGM-2 bullet kit. Culture dishes were precoated with 1% gelatin. For the labeling of CD8⁺ T-cells, CellTracker^TM green dye was used (Molecular Probes). The culture of EO771 and PyMT cells was described previously^{62 (link),63 (link)}. Other reagents include a JAK2 inhibitor (AG490), NFκΒ inhibitor (JSH23, Sigma), TNFα (Peprotech), IFNγ (EMD Millipore), GSK3484862 (Med Chem Express), and 5-Azacytidine (Sigma).

Kim D.J., Anandh S., Null J.L., Przanowski P., Bhatnagar S., Kumar P., Shelton S.E., Grundy E.E., Chiappinelli K.B., Kamm R.D., Barbie D.A, & Dudley A.C. (2023). Priming a vascular-selective cytokine response permits CD8+ T-cell entry into tumors. Nature Communications, 14, 2122.

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Isolation and Immunophenotyping of Tumor Cells

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Tumor tissues were minced and incubated with collagenase type II (1 mg/ml, Thermo Fisher) at 37°C for 40 min to obtain a single-cell suspension. For the staining of extracellular target proteins, 1 × 10⁶ cells were first incubated in a mixture of PBS, 1% FBS, and anti-CD16/32 antibody (BioLegend, #101301) to block non-specific binding and then labeled with the indicated antibodies at room temperature for 30 min. Fluorescence-activated cell sorting (FACS) was performed using a Beckman Coulter Gallios flow cytometer (USA), and the results were analyzed using FlowJo software version 10.0.7 (TreeStar). The following fluorochrome-coupled antibodies were used in the experiment: anti-CD45 (#103115), anti-CD206 (#141711), anti-CD3 (#100235), anti-CD4 (#100203), anti-CD8 (#100733), anti-NK1.1 (#108709), and anti-Gr1 (#108425) (all from BioLegend); and anti-CD11b (#12-0112), anti-F4/80 (#11-4801), anti-CD11c (25–0114), anti-MHCII (#17-5321), and anti-CD86 (#17-0862) (all from eBioscience) antibodies; the fixable viability dye eFluor 506 was from eBioscience (#65-0866).

Yu H., Bai Y., Qiu J., He X., Xiong J., Dai Q., Wang X., Li Y., Sheng H., Xin R., Jiang L., Li Q., Li D., Zhang H., Zhang L., Chen Q., Peng J., Hu X, & Zhang K. (2021). Pseudomonas aeruginosa PcrV Enhances the Nitric Oxide-Mediated Tumoricidal Activity of Tumor-Associated Macrophages via a TLR4/PI3K/AKT/mTOR-Glycolysis-Nitric Oxide Circuit. Frontiers in Oncology, 11, 736882.

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