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Anti cd11c n418

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Anti-CD11c (N418) is a monoclonal antibody that binds to the CD11c antigen. CD11c is a cluster of differentiation marker expressed on the surface of various immune cells, including dendritic cells, monocytes, and macrophages. This antibody can be used for the identification and isolation of these cell types.

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6 protocols using anti cd11c n418

1

Depletion and Immunophenotyping of Murine Hematopoietic Cells

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For sorting, FLs were depleted (Ter119+CD45+) using MACS Columns (Miltenyi Biotec; Cat# 130–042-401). Cell suspensions were stained for 20–30 min at 4°C in the dark with the following antibodies when appropriate: CD11b (M1/70), CD16/32 (93 or 2.4G2), CD19 (1D3), CD24 (M1/69), CD31 (MEC13.3 or 390), CD324 (DECMA-1), CD34 (RAM34), CD41 (MWReg30), CD45 (30-F11 or 104), CD54 (YN1/1.7.4), CD71 (C2), Gr1 (RB6-8C5), Il7ra (A7R34), Ki-67 (SolA15), c-Kit (2B8), Sca1 (D7), and Ter119 (obtained from SONY, BD Biosciences, Biolegend, or eBioscience). Biotinylated antibodies were detected by incubation for 15 min at 4°C in the dark with streptavidin. Antibodies to lineage markers included anti-Ter119 (TER-119), anti-Gr1 (RB6-8C5), anti-CD19 (6D5), anti-CD3 (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53–6.7), anti-NK1.1 (PK136), anti-Il7r (A7R34), anti-TCRβ (H57-597), anti-TCRγδ (GL3), and anti-CD11c (N418) obtained from SONY, BD Biosciences, Biolegend, or eBioscience. Stained cells were analyzed on a custom BD LSR Fortessa or BD FACSymphony and were sorted with a BD FACSAria III (BD Biosciences) according to the guidelines for the use of flow cytometry and cell sorting (Cossarizza et al., 2019 (link)). Data were analyzed with FlowJo software (BD Biosciences; v.10.5.3) or R packages as described in Bioinformatic analysis.
Soares-da-Silva F., Freyer L., Elsaid R., Burlen-Defranoux O., Iturri L., Sismeiro O., Pinto-do-Ó P., Gomez-Perdiguero E, & Cumano A. (2021). Yolk sac, but not hematopoietic stem cell–derived progenitors, sustain erythropoiesis throughout murine embryonic life. The Journal of Experimental Medicine, 218(4), e20201729.
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2

Immune Cell Phenotyping by Flow Cytometry

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Single cells were isolated from spleen and thymus and stained with monoclonal antibodies for immune cell phenotyping as previously described [28 (link)]. Briefly, after isolating the single cells, their non-specific binding was inhibited by blocking Fc receptors, and then the lymphocytes were stained with monoclonal antibodies; anti-CD4 (clone GK1.5), anti-CD8 (clone 53–6.7), anti-CD11b (clone M1.70) and anti-CD11c (N418) (BD biosciences, Franklin Lakes, NJ, USA). Lastly, the stained cells were fixed with 1% paraformaldehyde in phosphate-buffered solution (PBS). Approximately 10,000 cells from each sample were analyzed using a fluorescence-activated cell sorting (FACS) Caliver system (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).
Lee Y., Im S.A., Kim J., Lee S., Kwon J., Lee H., Kong H., Song Y., Shin E., Do S.G., Lee C.K, & Kim K. (2016). Modified Aloe Polysaccharide Restores Chronic Stress-Induced Immunosuppression in Mice. International Journal of Molecular Sciences, 17(10), 1660.
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3

Bone Marrow Transplantation: Assessing Donor Engraftment

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WT [CD45.1] and Trif−/− [CD45.2] mice were used so that donor and host cells could be distinguished from one another. Recipient mice were lethally irradiated using a Cesium irradiator (9.25 Gy over 12 min.), and were injected i.v. with 107 bone marrow cells. At 13 weeks post-irradiation, antibodies against CD45.1 (A20) and CD45.2 (104) were used to assess donor engraftment in T cells (anti-CD3), B cells (anti-CD19; 6D5), myeloid cells anti-CD11b (M1/70), and DCs and alveoloar macrophages (anti-CD11c; N418 and HL3), BD Biosciences, BioLegend and eBioscience, San Diego, CA. At least 85% reconstitution was observed for each cell population tested.
Hsia B.J., Whitehead G.S., Nakano K., Gowdy K.M., Thomas S.Y., Aloor J., Nakano H, & Cook D.N. (2014). Trif-dependent induction of Th17 immunity by lung dendritic cells. Mucosal immunology, 8(1), 186-197.
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4

Immunohistochemical Staining of Tumor Sections

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Tumors were harvested, bisected, and fixed using BD cytofix (diluted 1:3 in PBS) for 24 h at 4 °C. Fixed samples were washed twice with PBS and dehydrated with 30% sucrose for 24 h before embedding in OCT. 20 μm sections were prepared using a cryostat and blocked using a buffer containing 1% normal mouse serum, 1% bovine serum albumin, and 0.3% Triton-X 100 for 1 h. Sections were stained with directly conjugated antibodies for 8 h at RT or overnight at 4 °C in a dark humidified chamber, and imaged using a Leica SP8 microscope, as described (Gerner et al., 2012 (link)). The following antibodies were used for staining: anti-CD64 (X54–5/7.1, Biolegend), anti-CD11c (N418, BD), anti-CD45.1 (A20, BD Biosciences), anti-MHC-II (M5/114.15.2, Biolegend) conjugated in house with Dy396XL (Dyomics, 396XL-01A), anti-Fascin1 (55K-2, Santa Cruz Biotechnology), anti-CD31 (MEC 13.3, Biolegend) and anti-CD3e (17A2, Biolegend).
Di Pilato M., Kfuri-Rubens R., Pruessmann J.N., Ozga A.J., Messemaker M., Cadilha B.L., Sivakumar R., Cianciaruso C., Warner R.D., Marangoni F., Carrizosa E., Lesch S., Billingsley J., Perez-Ramos D., Zavala F., Rheinbay E., Luster A.D., Gerner M.Y., Kobold S., Pittet M.J, & Mempel T.R. (2021). CXCR6 Positions Cytotoxic T Cells to Receive Critical Survival Signals in the Tumor Microenvironment. Cell, 184(17), 4512-4530.e22.
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5

Fc Receptor Blocking for Immunophenotyping

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All staining reactions were performed at 4°C on cells first exposed to Fc receptor mAb (2.4G2) in order to reduce nonspecific binding. Anti CD11b (M1/70) and anti-CD11c (N418) were purchased from BD Biosciences-Pharmingen. Cells were analyzed with a BD LSRFortessa flow cytometer (BD) equipped with BD FACSDiva 7.0 software.
Moretti S., Bozza S., Oikonomou V., Renga G., Casagrande A., Iannitti R.G., Puccetti M., Garlanda C., Kim S., Li S., van de Veerdonk F.L., Dinarello C.A, & Romani L. (2014). IL-37 Inhibits Inflammasome Activation and Disease Severity in Murine Aspergillosis. PLoS Pathogens, 10(11), e1004462.
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6

Bone Marrow Transplantation: Assessing Donor Engraftment

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WT [CD45.1] and Trif−/− [CD45.2] mice were used so that donor and host cells could be distinguished from one another. Recipient mice were lethally irradiated using a Cesium irradiator (9.25 Gy over 12 min.), and were injected i.v. with 107 bone marrow cells. At 13 weeks post-irradiation, antibodies against CD45.1 (A20) and CD45.2 (104) were used to assess donor engraftment in T cells (anti-CD3), B cells (anti-CD19; 6D5), myeloid cells anti-CD11b (M1/70), and DCs and alveoloar macrophages (anti-CD11c; N418 and HL3), BD Biosciences, BioLegend and eBioscience, San Diego, CA. At least 85% reconstitution was observed for each cell population tested.
Hsia B.J., Whitehead G.S., Nakano K., Gowdy K.M., Thomas S.Y., Aloor J., Nakano H, & Cook D.N. (2014). Trif-dependent induction of Th17 immunity by lung dendritic cells. Mucosal immunology, 8(1), 186-197.
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