The cells were washed in FACS buffer (1% BSA, 0.01% sodium azide, 2.5 mM EDTA in sterile PBS; 650 g, 10 min, 4°C) and preincubated for 15 min with anti-CD16/CD32 Fc-block antibody (10 μg/mL; BioLegend, San Diego, CA, 1:50) in FACS buffer followed by a 30 min incubation with fluorescence dye-conjugated, anti-mouse antibodies for SiglecF (clone E50–2440, 1:400), CD11c (clone N418, 1:100), CD11b (clone M1/70, 1:100), Ly6G (clone 1A8, 1:50, clone 1A8, 1:400), Ly6C (clone HK1.4, 1:80/1:600), F4/80 (clone BM8, 1:100), CD62L (clone DREG-56, 1:80), CD66b (clone G10F5, 1:40), and corresponding isotype controls (all from BioLegend). Analysis was done in adherence to guidelines [25 (link)].
Cd11c clone n418
Manufactured by BioLegend
Sourced in United Kingdom, Switzerland, United States
CD11c (clone N418) is a cell surface marker that is expressed on dendritic cells, a type of antigen-presenting cell. It is involved in the adhesion and migration of dendritic cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b clone m1 70, by BioLegend (18 mentions)
Ly6c clone hk1, by BioLegend (11 mentions)
F4 80 clone bm8, by BioLegend (10 mentions)
Ly6g clone 1a8, by BioLegend (8 mentions)
Cd4 clone gk1, by BioLegend (8 mentions)
Cd45 clone 30 f11, by BioLegend (7 mentions)
Mhcii clone m5 114, by BioLegend (7 mentions)
Cd19 clone 6d5, by BioLegend (4 mentions)
Ly6g 1a8, by BioLegend (3 mentions)
Cd103 clone 2e7, by BioLegend (3 mentions)
Cd8 clone 53 6, by BioLegend (3 mentions)
Nk1.1 clone pk136, by BioLegend (3 mentions)
Cd11b clone m1 70, by Thermo Fisher Scientific (3 mentions)
Foxp3 clone fjk 16s, by Thermo Fisher Scientific (3 mentions)
Cd80 clone 16 10a1, by BioLegend (3 mentions)
Facsdiva software, by BD (3 mentions)
Cd45.2 clone 104, by BioLegend (3 mentions)
Cd45.1 clone a20, by BioLegend (3 mentions)
Cd64 clone x54 5 7, by BioLegend (3 mentions)
Facscanto 2, by BD (3 mentions)
33 protocols using cd11c clone n418
1
Comprehensive Immune Cell Phenotyping
2
Comprehensive Tumor Cell Phenotyping
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Totally, 1.5 million cells from tumors in mTmG mice were stained following the BD Cytofix/Cytoperm Fixation/Permeabilization microplate staining protocol (BD Biosciences), using the following antibodies: CD16/32 Fc Block (clone 2.4G2, BD Biosciences cat. 553142, 1:100), CD45 (clone 30-F11, BioLegend cat. 103127, 1:350), PD-L1 (clone 10F.9G2, BioLegend cat. 124313, 1:100), CD11b (clone M1/70, BioLegend cat. 101223, 1:100), CD11c (clone N418, BioLegend cat. 117323, 1:100). Cell lines were stained with PD-L1 (clone 10F.9G2, BioLegend cat. 124307, 1:100). Dead cells were excluded using Ghost Dye Violet 510 Dead Cell Stain Kit (Tonbo Biosciences) according to manufacturer protocol, and compensation was calibrated with single-color controls on Ultracomp eBeads (Invitrogen). Cells were quantified using a Gallios 561 cytometer (Beckman Coulter) and analyzed using Kaluza analysis software (Beckman Coulter) with fluorescence-minus-one controls to verify gating.
3
Immunostaining of Optically Cleared Tissues
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The following primary antibodies were used for immunostaining of optically cleared tissues: polyclonal rabbit anti‐α‐smooth muscle actin (SMA) (Abcam; ab5694; 1 : 300); and from (BioLegend UK Ltd, London, UK): rat anti‐CD45 clone 30‐F11 (103102; 1 : 300), CD11c clone N418 (117302; 1 : 200) and MHCII I‐A/I‐E clone M5/114.15.2 (107601; 1 : 300). The following macrophage markers were found to be unreliable with the tissue clearing protocols used: rat anti‐F4/80 (clone BM8), rat anti‐CD11b (clone M1/70), rat anti‐CD68 (clone FA‐11) (all from BioLegend) and rat anti‐F4/80 (clone Cl:A3‐1; from AbD Serotec, Kidlington, UK). The following secondary antibodies (all used at 1 : 500) were purchased from Invitrogen: goat anti‐rabbit Alexa Fluor 488 (A11008), goat anti‐rat Alexa Fluor 647 (A21247) and goat anti‐rat Cy3 (A10522); and from (Jackson ImmunoResearch, Ely, UK): goat anti‐Armenian hamster Cy3 (127‐165‐160).
For 2D analysis, SMA expression was detected using a mouse anti‐human SMA primary antibody (clone 1A4, Agilent; M0851) with a peroxidase‐conjugated ImmPRESS anti‐mouse IgG polymer detection kit (Vector Laboratories Ltd; MP‐7402, Peterborough, UK) using standard development with DAB. Mouse IgG1 was used for species‐ and isotype‐matched control (Agilent; X0931).
For 2D analysis, SMA expression was detected using a mouse anti‐human SMA primary antibody (clone 1A4, Agilent; M0851) with a peroxidase‐conjugated ImmPRESS anti‐mouse IgG polymer detection kit (Vector Laboratories Ltd; MP‐7402, Peterborough, UK) using standard development with DAB. Mouse IgG1 was used for species‐ and isotype‐matched control (Agilent; X0931).
4
Multicolor Flow Cytometry Immunophenotyping
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For flow cytometry or cell sorting, cells were transferred to 15-mL tubes and washed in cold buffer (2% FCS, 2 mM EDTA in PBS). Before antibody labeling, cells were incubated with Fc-Block (1:200, anti-CD16/32 antibody; 2.4G2, BD Pharmingen) for 10 min at 4°C and washed. Cells were stained in buffer with (1:100) fluorescently labeled antibodies against CD11b (clone M1/70, BD Pharmingen), CD11c (clone N418, Biolegend), CD115 (clone AFS98, eBioscience), CD19 (clone 1D3, BD Pharmingen), and Ly-6G (1A8, Biolegend) for 30 min at 4°C in the dark. 7-AAD (BD Pharmingen) was added before measurement to exclude dead cells. Analysis was performed on LSRFortessa and sorting on FACSAria II or III (BD Pharmingen). Unstained, empty vector-transduced cells or fluorescence-minus-one staining setups served as controls.
5
Detecting ERV Envelope on Lymphocytes
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ERV envelope on lymphocytes was detected as previously described (42 (link); b)using an anti-MLV envelope antibody clone 83A25 (kindly provided by Leonard Evans, NIH) (41 (link)). The following antibodies were used for staining of vaginal dendritic cells: Fixable Aqua Dead Cell Stain Kit (Thermo Fisher), CD45 (clone 30-F11, BioLegend), CD11b (clone M1/70, BioLegend), and CD11c (clone N418, BioLegend). All cells were stained in 1% BSA PBS and incubated on ice for 15 to 20 minutes. Cells were acquired on BD LSRII cytometer and analyzed by FlowJo software v8.8.7 (Tree Star, Inc.).
6
Multiparameter Flow Cytometry Panel
7
Tumor Cell and Immune Cell Profiling
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As described previously35 , subcutaneous tumours with KP1.9 cells were harvested from C57BL/6 flanks 3 weeks after implantation, minced, and shaken at 600 r.p.m. with 0.2 mg ml−1 collagenase type I (Worthington Biochemical Corporation) in RPMI-1640 for 30 min at 37 °C. Digested samples were filtered (70 μm BD Falcon strainer); washed in PBS with 0.5% BSA and 2 mM EDTA; incubated with Fc-block (TruStain fcX anti-mouse CD16/32; clone 93; Biolegend) for 15 min at 4 °C; and labelled with antibodies as indicated for 45 min at 4 °C. Flow cytometry (LSRII, BD Biosciences) labelled tumour cells (CD45− EpCAM+), TAM (CD45+ CD11b+ Ly6C- Lin- CD11c+ F4/80+), lymphocyte-like cells (CD45+ CD11b- Lin+), along with CD45- EpCAM- host-cell populations. Antibodies included EpCAM (clone G8.8; eBioscience); CD45 (clone 30-F11; Biolegend), F4/80 (clone BM8; Biolegend), CD11c (clone N418; Biolegend), Ly6C (clone HK1.4; Biolegend); and CD11b (clone M1/70; BD Biosciences). The lineage (Lin) antibody mix contained anti-CD90.2 (clone 53–2.1), anti-B220 (clone RA3-6B2), anti-NK1.1 (clone PK136), anti-CD49b (clone DX5), anti-Ter119 (cloneTER-119) and anti-Ly6G (clone 1A8) (all BD Biosciences). 7-aminoactinomycin D (7-AAD, Sigma Aldrich) excluded dead cells. VT680 fluorescence was directly assessed using the LSRII flow cytometer, FlowJo v.8.8.7 (Tree Star, Inc.) and MATLAB.
8
Identification of Lung ILC2 Cells
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Lung ILC2 cell identification was performed as described previously (47 (link)). Lung tissues were digested in 8 mL RPMI 1640 containing liberase (50 μg/mL) and DNase I (1 μg/mL) for approximately 40 minutes at 37°C. Cell suspensions were filtered through 70 μm cell strainers and washed once with RPMI 1640. For ILC2 cell identification, total lung cell suspensions were blocked with 2.4G2 antibodies and stained with lineage cocktail mAbs: CD3ε (clone 145-2C11) (BioLegend, 100304), CD4 (clone GK1.5) (BioLegend, 100404), CD8α (clone 53-6.7) (Tonbo Biosciences, 30-0081-U500), CD11c (clone N418) (BioLegend, 117304), FceRIα (clone MAR-1) (BioLegend, 134304), NK1.1 (clone PK136) (BioLegend, 108704), CD19 (clone 6D5) (BioLegend, 115504), TER119 (clone TER-119) (BioLegend, 116204), CD5 (clone 53-7.3) (BioLegend, 100604), F4/80 (clone BM8.1) (Tonbo Biosciences, 30-4801-U500), Ly6G (clone RB6-8C5) (Tonbo Biosciences, 30-5931-U500), APC-conjugated streptavidin (BioLegend, 405207), PE-conjugated T1/ST2 (clone DIH9) (BioLegend, 145304), PerCP-Cy5.5-conjugated CD25 (clone PC61) (BioLegend, 102030), V450-conjugated Sca-1 (clone D7) (BD Biosciences, 560653), PE-Cy7-conjugated KLRG1 (clone 2F1/KLRG1) (BioLegend, 138416), APC-Cy7-conjugated CD45, and Fixable Viability Dye eFluor 506.
9
Isolation and Characterization of Lung Immune Cells
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BALF was recovered from mice at the specified timepoint in 1 ml sterile PBS containing 5 mM EDTA as previously described [90 (link)]. Cells were surface stained 30 min in cold FACS buffer (PBS with 2.5% FBS, 5 mM EDTA) with Siglec-F (clone E50-2440, Biolegend), CD11c (clone N418, Biolegend), Ly6G (clone 1A8, BD Biosciences), Ly6C (clone AL-21, BD Biosciences, Allschwil, Switzerland), CD11b (clone M1/70, Biolegend), CD45.1 (clone A20, BD Biosciences), CD45.2 (clone 104, BD Biosciences).
For intracellular staining of TNF (clone MP6-XT22, Biolegend), mice were injected i.p. with 50 μl of 5 mg/ml Brefeldin A in EtOH (diluted with 100 μl PBS) 3 hours prior to taking BALF. Lavage was performed with 1 ml PBS 5mM EDTA containing 5 μg/ml Brefeldin A, and was immediately placed on ice. After surface stain, cells were washed with FACS buffer and fixed, permeabilized and stained using the BD Biosciences Cytofix/Cytoperm Kit according to the manufacturer's instructions. Data were acquired on an LSRII (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, OR). An Aria III instrument (BD Biosciences) was used for cell sorting.
For intracellular staining of TNF (clone MP6-XT22, Biolegend), mice were injected i.p. with 50 μl of 5 mg/ml Brefeldin A in EtOH (diluted with 100 μl PBS) 3 hours prior to taking BALF. Lavage was performed with 1 ml PBS 5mM EDTA containing 5 μg/ml Brefeldin A, and was immediately placed on ice. After surface stain, cells were washed with FACS buffer and fixed, permeabilized and stained using the BD Biosciences Cytofix/Cytoperm Kit according to the manufacturer's instructions. Data were acquired on an LSRII (BD Biosciences) and analyzed with FlowJo software (TreeStar, Ashland, OR). An Aria III instrument (BD Biosciences) was used for cell sorting.
10
Multiparametric Flow Cytometry of Liver and Spleen
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For flow cytometric analysis, liver and spleen single-cell suspensions were stained using the following antibodies: Cd11c, clone N418 (Biolegend, San Diego, CA, 117339); Ly6c, clone HK1.4 (Biolegend, 128035); MHCII, clone M5/114.15.2 (eBioscience, Waltham, MA, 48-5321-82); Cd45, clone 30-F11 (eBioscience, 56-0451-82); Cd11b, clone M1/70 (eBioscience, 47-0112-82); Cd64, clone X54-5/7.1 (BD Biosciences, Franklin Lakes, NJ, 741024); Ly6g, clone 1A8 (BD Biosciences, 560601); Fc block Cd16/Cd32, clone 2.4G2 (BD Biosciences, 553142); Ly6g, RB6-8c5 (Tonbo, San Diego, CA, 60-5931); Ghost (Tonbo, 13-0870-T100); Flow cytometric data was acquired on the BD LSRII Fortessa X20 and analyzed using FlowJo (Franklin Lakes, NJ).
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