Antibodies employed in flow cytometric analysis were obtained from various commercial sources: anti-CD1d (Biolegend, 1B1), anti-αGalcer:CD1d (eBioscience, L363), CD86 (BD Bioscience, GL1), TCRαβ (eBioscience,IP26), F4/80 (eBioscience, BM8), IL17-A (Biolegend,TC11-18H10.1), NK1.1 (Biolegend, PK136), and CD3 (Biolegend, 17A2). Cells were blocked with anti-CD16/32 antibody (Biolegend) for 15 min before incubation with fluorescently labeled antibodies at a concentration of 2 μg/ml for 45 min on ice. Stained cells were washed once with FACS buffer and analyzed by FACSan (Becton Dickinson). For internal staining, cells stained with surface makers were fixed in 2% PFA Fixation buffer (eBioscience) at 4°C overnight, followed by 3 washes with permeabilization buffer (R&D) and incubation with permeabilization buffer for 20 min on ice before staining with internal antibody. Stained cells were washed once with permeabilization buffer and suspended in PBS for analysis. Flow cytometry data were processed with FlowJo software (Tree Star, inc.,).
Tc11 18h10
Manufactured by BioLegend
TC11-18H10.1 is a mouse monoclonal antibody that recognizes the CD11c antigen. CD11c is a 150 kDa integrin alpha X chain expressed on the surface of dendritic cells, macrophages, and other myeloid cells. This antibody can be used for flow cytometry and other immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Af6 120, by BioLegend (3 mentions)
M1 70, by BD (3 mentions)
Cd4 gk1, by BioLegend (3 mentions)
Facscanto 2, by BD (2 mentions)
Rm4 5, by Thermo Fisher Scientific (2 mentions)
Ebio13a, by BioLegend (2 mentions)
Live dead blue or aqua, by Thermo Fisher Scientific (2 mentions)
X54 5 7, by BioLegend (2 mentions)
E50 2440, by Thermo Fisher Scientific (2 mentions)
H57 597, by Thermo Fisher Scientific (2 mentions)
Rb6 8c5, by BioLegend (2 mentions)
Brefeldin a, by Thermo Fisher Scientific (2 mentions)
Mp6 xt22, by BioLegend (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Golgiplug, by BD (2 mentions)
Anti cd16 32 antibody, by BioLegend (1 mentions)
F4 80, by Thermo Fisher Scientific (1 mentions)
Facsan, by BD (1 mentions)
Permeabilization buffer, by R&D Systems (1 mentions)
16 protocols using tc11 18h10
1
Flow Cytometric Immune Cell Analysis
2
Multiparameter Flow Cytometry Analysis
3
Multi-Parametric Flow Cytometry Analysis of Murine T Cell Subsets
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Fluorescence-conjugated antibodies for CD4 (RM4-5), IFN-γ, IL-17A (TC11-18H10.1), CD45RB (MEM-55), and CD90.1 (H1.2F3) were purchased from Biolegend and CD25 (PC61.5) and FoxP3 (FJK-16s) were from eBioscience. For FACs staining, 0.5 – 1 × 106 cells were collected and surface stained followed by intracellular staining after fixation and permeabilization according the manufacture's instruction (BD Bioscience). For intracellular cytokine staining, lymphocytes were stimulated for 4 h with 50 ng/mL of PMA (phorbol 12-myristate 13-acetate) and 1 mM ionomycin in the presence of brefeldin A. Stained cells were analyzed on the Canto II ™ (BD biosciences) and FACS data were analyzed with FlowJo software (TreeStar). For naïve T cell sorting, CD4+ T cells were enriched by MACs and then stained with fluorescence-conjugated antibodies and CD4+CD25−CD45RBhi naïve cells were sorted on the Moflow cell sorter (Dako cytomation, Beckman coulter) by the flow facility of University of North Carolina at Chapel Hill or the BD FACSAria™ III sorter in Xuzhou Medical University.
4
Cytokine profiling of stimulated cells
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Sorted cell populations were stimulated with 1 µg/ml ionomycin and 50 ng/ml PMA for 5 h at 37°C in the presence of Brefeldin A (eBioscience). Cells were labeled with Live/Dead fixable violet dead cell stain kit (Invitrogen) and then fixed with 4% paraformaldehyde according to the manufacturer’s protocol. Cells were permeabilized and stained in Perm/Wash buffer (BD) using Ab against IFN-γ (XMG1.2) and IL-17A (TC11-18H10.1; BioLegend) for analysis by flow cytometry.
5
Detailed Immune Cell Profiling
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For surface staining, anti-PD-L1 PE (1:200, J43, BD Biosciences #551892) was stained for 30 min on ice and dead cell was stained with Propidium iodide (1:2000, DOJINDO #341-07881) before FACS analysis. For intracellular staining, dead cells were first stained with Fixable Viability Dye eFluor 780 (1:1000, eBioscience #65-0865-14) for 10 min. For cytokine staining, sample preparation was conducted with 4% PFA for 10 min at 4 °C and incubated with permeabilization buffer for 10 min on ice (50 mM NaCl, 5 mM EDTA, 0.5% Triton-X). After incubating with blocking buffer for 15 min on ice (0.3% BSA), the cells were stained with anti-TNFα PE (1:200, MP6-XT22, Biolegend# 506306), anti-IFNγ FITC (1:50, XMG1.2, BD Biosciences #554411), anti-IL-2 APC (1:1000, JES6-5H4, BD Biosciences #554429), anti-IL-4 BV421 (1:200, 11B11, BD Biosciences #562915), anti-IL-17A BV421 (1:200, TC11-18H10.1, Biolegend#506925), anti-IL-17F APC (1:200, O79-289, BD Biosciences #561630), or for 30 min in the dark. Cell proliferation and cell cycle were analyzed with Proliferation dye e670 (Invitrogen #65-0840-90) or BrdU Flow kit (BD Biosciences #557891) as according to the manufacture’s protocol. Flow cytometric data were analyzed after removal of dead cells and doublets cells with Flowjo software (version 10.4).
6
Quantitative Cytokine ELISA Assay
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Primary antibodies (αIFN-γ; R4-6A2; BioLegend; αIL-17A; TC11-18H10.1; BioLegend) were diluted in ELISA Coating Buffer (BioLegend) and coated on ELISA plates overnight at 4°C. Coated plates were blocked with PBS containing 1% BSA for 2 h. Then samples and appropriate standards were loaded in duplicate, diluted in blocking buffer, and incubated overnight. Detection antibodies (αIFN-γ; XMG1.2; BioLegend; αIL-17A; TC11-8H4; BioLegend) were used according to manufacturers’ instructions. Plates were washed extensively in between steps with PBS and 0.05% Tween-20, and were developed using the o-Phenylenediamine colorimetric assay and read at 490 nm using a iMark microplate reader (Bio-Rad).
7
Isolation and Characterization of Murine Immune Cells
8
Isolation and Stimulation of Lung-Derived White Blood Cells
9
Comprehensive Murine Immune Cell Profiling
10
Antibody Panel for T-cell Analysis
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The following antibodies were used for flow cytometry analysis and fluorescence-activated cell sorting (FACS): Anti-CD3 (17A2, Biolegend, 1:400), anti-CD4 (GK1.5, Biolegend, 1:100 or 1:400), anti-CD8 (53–6.7, Biolegend, 1:400), anti-CD25 (PC61, Biolegend, 1:400), anti-CD44 (IM7, Biolegend, 1:400), anti-CD62L (MEL-14, Biolegend, 1:400), anti-IL-17A (TC11-18H10.1, Biolegend, 1:100), anti-IFN-γ (XMG1.2, Biolegend, 1:100), anti-GM-CSF (MP1-22E9, BD, 1:100), anti-RORγt (Q31-378, BD, 1:50), anti-Foxp3 (150D, Biolegend, 1:50), anti-T-bet (4B10, Biolegend, 1:50) and anti-TCRVβ8.1/8.2 (KJ16-133.18, Biolegend, 1:20). For immunoblot analyses, anti-JunB (C37F9, Cell Signaling Technology, 1:2,000), anti-RORγt (AFKJS-9, eBioscience, 1:400) and anti-GAPDH (3H12, MBL, 1:2,000) were used. For ChIP analyses, anti-JunB (210, Santa Cruz, 2 μg per ChIP), anti-BATF (WW8, Santa Cruz, 2 μg per ChIP), anti-IRF4 (M-17, Santa Cruz, 2 μg per ChIP), anti-STAT3 (c-20, Santa Cruz, 2 μg per ChIP) were used.
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