Freshly isolated PBMCs or TILs (1 × 106) were washed and incubated with fluorophore-conjugated monoclonal antibodies including Alexa Fluor 750-anti-CD45, Alexa Fluor 700-anti-CD3, BV421-anti-TCRγδ, FITC-anti-Vδ1, and PE-anti-Vδ2 (all from Biolegend, San Jose, CA, USA) for 20 min at room temperature in the dark. For intracellular cytokine detection, PBMCs or TILs (1 × 106) were resuspended in RPMI 1640 medium supplemented with 10% FBS (Gibco) and stimulated with phorbol-12-myristate 13-acetate (50 ng/ml), ionomycin (1 μg/ml), and brefeldin (1 μg/ml) (all from Biogems, Rocky Hill, NJ, USA) for 5 h in 5% CO2 atmosphere at 37 °C. Cells were then washed, fixed, permeabilized, and stained with APC-anti-IL-17A, and APC/cy7-anti-IFN-γ (Biolegend, San Jose, CA, USA) according to the manufacturer’s protocol. Fluorescence data were collected on a FACS Aria II (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software (Tree Star, Ashland, OR, US).
Anti il 17a apc
Manufactured by BioLegend
Sourced in United States
Anti-IL-17A APC is a fluorochrome-conjugated antibody that binds to the interleukin-17A (IL-17A) cytokine. It can be used for the detection and quantification of IL-17A-expressing cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti ifn γ pe, by BioLegend (3 mentions)
Anti il 4 pe, by BD (2 mentions)
Fixable viability stain 510 antibody, by BD (2 mentions)
Alexa fluor 700 anti cd3, by BioLegend (1 mentions)
Phorbol 12 myristate 13 acetate, by Biogems (1 mentions)
Ionomycin, by Biogems (1 mentions)
Alexa fluor 750 anti cd45, by BioLegend (1 mentions)
Fitc anti vδ1, by BioLegend (1 mentions)
Pe anti vδ2, by BioLegend (1 mentions)
Bv421 anti tcrγδ, by BioLegend (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
Aluminum hydroxide gel, by Merck Group (1 mentions)
Bicinchoninic acid (bca), by Beyotime (1 mentions)
Anti gr1, by BioLegend (1 mentions)
Max 1320, by Buxco Electronics (1 mentions)
Pan cytokeratin, by Santa Cruz Biotechnology (1 mentions)
Mbd2 antibody, by Abcam (1 mentions)
Anti ecp, by Biorbyt (1 mentions)
8 protocols using anti il 17a apc
1
Immunophenotyping of γδ T Cells
2
Asthma Mouse Model Reagents
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GREER laboratories supplied HDM; OVA, LPS, and aluminum hydroxide gel from Sigma; mice spirometer (MAX 1320) from Buxco®, USA; Beijing Zhongshidichuang Science and Technology Development Co. Ltd. supplied animal asthma inducing instrument (YLS-8A); centrifuge (5810 R) from Eppendorf®, Germany; image analyzer (Leica Application Suite V4) from Leica, Germany; and Olympus, Japan (Leica®) supplied optical microscope (DMI3000B); 150-mesh cell sieve (Biosharp, BS-100-XBS, China); bronchial epithelial growth medium (Procell, CM-M007, China); DAPI and cytokeratin specific monoclonal antibody (pan-Cytokeratin, Santa Cruz, sc-8018, USA); leukocyte activation cocktail (550583, BD Biosciences, USA); cell viability marker (Fixable Viability Stain 510 antibody, BD Pharmingen); PE-anti-IL-4 (BD Pharmingen); APC-anti-IL-17A (Biolegend); Lipofectamine 3000 (Invitrogen, USA); bicinchoninic acid (BCA), Beyotime, Shanghai, China; DHT (B8214) and 17β-estradiol (C4348) APExBIO, USA; MBD2 antibody (Abcam, Cambridge, USA); eosinophil antibody (anti-ECP, Biorbyt, Cambridge, UK); neutrophil antibody (anti-Gr-1, Biolegend, San Diego, USA); GATA3 (10417-1-AP) and β actin (60008-1-Ig) Proteintech, USA; RORγt (EPR20006) abcam, USA.
3
Flow Cytometric Analysis of T Cell Activation
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CD4+ T cells were stimulated with 2 μL/ml (1 × 106 cells/ml) leukocyte activation cocktail (550583, BD Biosciences, United States ), and cultured at 37°C with 5% CO2 for 6 h, then collected for flow cytometry analysis. After a 6-h incubation, cells were stained with a marker of cell viability (Fixable Viability Stain 510 antibody, BD Pharmingen) for 15 min at room temperature in the dark. Then, cells were stained for surface markers with FITC-anti-CD4 antibody (Biolegend) followed by fixation and permeabilization using the Cytofix/Cytoperm Soln Kit (BD Pharmingen) for 30 min at 4°C in the dark. After washing with permeabilization buffer, cells were stained for intracellular markers with APC-anti-IL-17 A (Biolegend) and PE-anti-IL-4 (BD Pharmingen) antibodies in permeabilization buffer for 30 min at 4°C in the dark. Isotype controls were employed in the control group. Flow cytometry was performed, and data were analyzed using FACS CantoⅡ(Becton Dickinson) and FlowJo version X software.
4
Multi-color FACS Analysis of Murine Immune Cells
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Mouse colons and lymph nodes were processed for single cell suspension as previously published [44 (link)] and multi-color staining performed according to standard FACS staining protocols. Briefly, tissues were treated with collagenase (I, II, and IV, Sigma Aldrich, St. Louis, MO) and dispersed twice using the gentleMACs tissue dissociator (Miltenyi Biotech, Cologne, Germany). Cell suspensions were incubated in complete RPMI media for 24 hours before staining for flow cytometry. Multi-color staining was performed according to standard surface and intracellular FACS staining Biolegend protocols (Biolegend, San Diego, CA). Antibodies used in this study were anti-Ly6G-APC (clone1A8, Biolegend), anti-F4/80-PCP (PerCP/Cy5.5), (Biolegend, BM8), anti-IL-12p35-APC (eBioscience, 4010p35), anti-IL-10-FITC (Biolegend, JES5-16E3), anti-NK1.1-PCP (eBioscience, PK136), anti-CD3-FITC (Biolegend, 145-2C11), anti-CD4-PCP (Biolegend, GK1.5), anti-CD8-PCP (Biolegend, 53-6.7), anti-Tbet-FITC (Biolegend, 4B10), anti-IFNγ-PE (Biolegend, XMG1.2), anti-IL-17A-APC (Biolegend, TC11-18H10.1), and anti-IL-4-FITC (eBioscience, 11-7042-82). All samples were analyzed on a Guava easyCyte 8HT flow cytometer (EMD Millipore, Bellerica, MA, USA), and analyzed using FCS Express software (DeNovo Software, Los Angeles, CA, USA).
5
Cytokine Expression in Lymph Node Cells
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Draining lymph node (DLN) cells (106/ml) were incubated ± 50 ng/ml PMA plus 500 ng/ml ionomycin for 1 h before addition of 10 μg/ml Brefeldin A (Sigma–Aldrich, UK) for a further 5 h at 37 °C with 5% CO2. Live cells were discriminated by the LIVE/DEAD fixable aqua dye (Invitrogen) and phenotypic markers were labelled using anti-CD4-PerCP, anti-CD8-FITC or anti-γδ-PE (BioLegend) antibodies before the cells were fixed and permeabilised using BioLegend protocols. Cells were then labelled using anti-IFNγ-Pacific Blue or anti-IL-17A-APC (BioLegend) antibodies for 30 min prior to flow cytometry and gated according to appropriate isotype controls as described previously [7] (link). IL-12p40 and IL-17 levels in serum or DLN, bmM and peritoneal exudate cell (PEC) supernatants were detected by ELISA using kits from BioLegend as described previously [7] (link) whilst levels of IL-1β were determined by ELISA using kits from eBioscience according to the manufacturer's recommendations.
6
Induction and Analysis of Th17 Cells
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Naive CD4+ T cell isolation kit was purchased from Miltenyi Biotec (Cologne, Germany). Anti-CD3/CD28 coated beads were purchased from Gibco (New York, USA). RhIL-2, rhTGF-β, rmIL-6, and rmIL-12 were purchased from R&D Systems (Minneapolis, MN). Anti-TNFR1 PE, anti-TNFR2 PE, anti-Thy1.1 PercP/Cy5.5, anti-LAP APC, anti-PD-1 APC, anti-Nrp-1APC anti-GITR PE, anti- Ki-67 PE, anti-CD4 PercP/Cy5.5, anti-IFN-γ PE, anti-IL-17A APC, rmTNFα, anti-IL-4 antibody, anti-IFN-γ antibody, and CFSE were purchased from Biolegend (San Diego, CA). PMA, ionomycin, and cyclosporin A (CsA) were purchased from Calbiochem-EMD Millipore (Dormstadt, Germany). CFA was purchased from Sigma-Aldrich. Incomplete Freund's Adjuvant (IFA, Difco, MI, USA), killed Mycobacterium tuberculosis (strain H37Ra; Difco), and myelin oligodendrocyte glycoprotein (MOG, 35–55 peptide, (AnaSpec Inc, Fremont, CA) were also purchased.
7
Multicolor Flow Cytometry Immunophenotyping
8
Th17 and IL-23R Cell Profiling
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Th17 cells and IL-23R were characterized using flow cytometry. Colon tissue-derived lymphocytes from mice were stimulated by adding 10 ng/mL of PMA [Sigma, USA], 1 μM of ionomycin [MCE, USA], and Golgistop [BD Biosciences, USA] before the experiment. Subsequently, the cells were washed with phosphate-buffered saline (PBS).
The lymphocytes were stained using anti-CD4 FITC [Biolegend, USA] and anti-IL-17 PE antibodies [Biolegend, USA] to identify Th17 cells. Further, antibodies including anti-IFN-γ PE [Biolegend, USA], anti-IL-17A APC [Biolegend, USA], and anti-IL-23R PE [Biolegend, USA] were separately added to stain lymphocytes for specific markers. Flow cytometry was utilized to determine the percentages of CD4 + IL-17A + (Th17) and CD4 + IL-17A + IL-23R + (IL-23R) cells. Analysis of the obtained data was performed using FlowJo V.10 software.
The lymphocytes were stained using anti-CD4 FITC [Biolegend, USA] and anti-IL-17 PE antibodies [Biolegend, USA] to identify Th17 cells. Further, antibodies including anti-IFN-γ PE [Biolegend, USA], anti-IL-17A APC [Biolegend, USA], and anti-IL-23R PE [Biolegend, USA] were separately added to stain lymphocytes for specific markers. Flow cytometry was utilized to determine the percentages of CD4 + IL-17A + (Th17) and CD4 + IL-17A + IL-23R + (IL-23R) cells. Analysis of the obtained data was performed using FlowJo V.10 software.
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