Il17a apc

Manufactured by BioLegend

Sourced in United States

IL17A-APC is a fluorescently-labeled antibody that binds to the interleukin-17A (IL-17A) protein. It is commonly used in flow cytometry and other immunoassays to detect and quantify the expression of IL-17A in cell samples.

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6 protocols using il17a apc

Comprehensive Immune Cell Profiling

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The heparinised whole blood was withdrawn from the mouse orbital sinus. Red blood cells were isolated with Easy Lyse Solution (10 mL; Leinco Technologies, USA), and then stained with Foxp3-PE, CD4-FITC, IL17A-APC, IFN-γ-Pacific Blue, IL4-PE, CD3-FITC and CD8-Pacific Blue (BioLegend, CA, USA). For graft analysis, the graft samples were homogenised and digested with collagenase (0.125%; Sigma, Missouri, USA) in a shaking water bath at 37 °C for 30 min. After centrifugation (800×g, 3 min) and resuspension, the cells were stained with the following monoclonal antibodies: CD11b-Pacific Blue, CD11c-PE/Cy7, Foxp3-PE, F4/80-PE, CD206-FITC CD4-FITC, IL17A-APC, IFN-γ-Pacific Blue, IL4-PE, CD3-FITC, and CD8-Pacific Blue (BioLegend, CA, USA). An irrelevant control monoclonal antibody was included for each fluorochrome. Finally, the cells were analysed using a BD LSR-II flow cytometer (Becton Dickinson, CA, USA). Cell Quest software (Becton Dickinson, CA, USA) was employed for data acquisition and analysis. A gate was set to exclude 99.9% of the isotype control-labelled cells.

Liu K., He Y., Yao Y., Zhang Y., Cai Z., Ru J., Zhang X., Jin X., Xu M., Li Y., Ma Q., Gao J, & Lu F. (2021). Methoxy polyethylene glycol modification promotes adipogenesis by inducing the production of regulatory T cells in xenogeneic acellular adipose matrix. Materials Today Bio, 12, 100161.

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Flow Cytometry Analysis of Splenic and Blood Cells

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Flow cytometry analysis was performed using a Guava 8HT (EMD Millipore Corporation, Billerica, MA USA). Splenic and blood cells from control and tolerant mice were collected and labeled with antimouse antibodies (BioLegend, San Diego, CA) for cell surface markers: CD4-PE and CD25- APC/Cy7. After cell membrane permeabilization with saponin, cells were intracellular labeled with antimouse antibodies: FOXP3-PE/Cy7, IL-17a- APC and IL-6-FITC (BioLegend).

Andrade M.M., Ariga S.S., Barbeiro D.F., Barbeiro H.V., Pimentel R.N., Petroni R.C, & Soriano F.G. (2019). Endotoxin tolerance modulates TREG and TH17 lymphocytes protecting septic mice. Oncotarget, 10(37), 3451-3461.

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Isolation and Analysis of Murine Immune Cells

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Spleen, MLN and kidney were collected and mashed in 70-μm cell strainers with C10 media (RPMI 1640, 10% fetal bovine serum, 1 mM sodium pyruvate, 1% 100 MEM non-essential amino acids, 10 mM HEPES, 55 μM 2-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin, all from Life Technologies, Grand Island, NY). For splenocytes, red blood cells were lysed with RBC lysis buffer (eBioscience, San Diego, CA). To isolate intestinal epithelial cells (IECs), the entire intestine including small intestine and colon was opened longitudinally and cut into pieces. The pieces were incubated twice in EDTA-DTT solution and intensively vortexed to harvest IEC-enriched fractions. For surface marker staining, cells were blocked with anti-mouse CD16/32 (eBioscience), stained with fluorochrome-conjugated antibodies, and analyzed with Attune NxT flow cytometer (Thermo Scientific). For intracellular staining, Foxp3 Fixation/Permeabilization kit (eBioscience) was used. Anti-mouse antibodies used in this study include: CD3-APC-eFluor 780, CD8-PE-Cy7, CD4-PerCP-Cy5.5, RORγT-PE, CD3e-biotin (eBioscience); CD45-APC-Cy7, IL-10-BV421, IL-17A-APC, CD49b-biotin, CD19-biotin (Biolegend, San Diego, CA); Biotin-FITC (MACS). Flow cytometry data were analyzed with FlowJo.

Mu Q., Tavella V.J., Kirby J.L., Cecere T.E., Chung M., Lee J., Li S., Ahmed S.A., Eden K., Allen I.C., Reilly C.M, & Luo X.M. (2017). Antibiotics ameliorate lupus-like symptoms in mice. Scientific Reports, 7, 13675.

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Profiling T Cell Subsets in PBMC Responses

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Live conidia and hyphal fragments stimulated PBMCs for 6 h and inactivated ones for 24 h. At the last 6 h of incubation, PMA (25 ng/mL), ionomycin (1 μg/mL) and brefeldin A (10 μg/mL) were added. Next, cells were collected and washed with PBS, followed by extracellular staining with anti-human CD3 PE-Cy7, CD4 APC-Cy7 and CD8 PE-Cy5.5 for 15 min. After that, PBMCs were centrifuged and resuspended in Fixation/permeabilisation reagents (Biogems, USA) for 30 min to perform the subsequent intracellular cytokine staining with anti-human IL-4 R-PE, IL-17a APC and IFN-γ Alexa Flour™ 700 (all antibodies: BioLegend, USA). Cell populations were analysed through flow cytometry. T lymphocyte was defined as CD3⁺ cells, and T cell subsets were defined as Th1: CD4⁺ IFN-γ⁺, Th2: CD4⁺ IL-4⁺ and Th17: CD4⁺ IL-17a ⁺. CD4 (%) = CD3⁺CD4⁺/CD3⁺, CD8 (%) = CD³⁺CD⁸⁺/CD³⁺, Th1 (%) = CD3⁺CD4⁺IFN-γ⁺/CD3⁺CD4⁺, Th2 (%) = CD3⁺CD4⁺IL-4⁺/ CD³⁺CD⁴⁺, Th17 (%) = CD3⁺CD4⁺IL-17a⁺/ CD3⁺CD4⁺

Luo Y., Liu F., Deng L., Xu J., Kong Q., Shi Y, & Sang H. (2022). Innate and Adaptive Immune Responses Induced by Aspergillus fumigatus Conidia and Hyphae. Current Microbiology, 80(1), 28.

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Murine Immune Cell Characterization

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The reagents and antibodies used in this study were referenced from our previous reports^{28 (link)–30 (link),32 (link),60 (link)}. Lipopolysaccharide (LPS) from Escherichia coli (LPS O55:B5, L2880) was purchased from Sigma-Aldrich (MO, USA). Pam3csk4, CpG-ODNs and cGAMP (cyclic guanosine monophosphate–adenosine monophosphate) were purchased from Invivogen (CA, USA). Murine GM-CSF (granulocyte–macrophage colony-stimulating factor) was purchased from Peprotech (Rocky Hill, NJ, USA). The anti-mouse TLR7 mAb A94B10 and the anti-TLR9 mAb NaR9 were purified from ascitic fluid, as reported previously^{29 (link),30 (link)}. Streptavidin–phycoerythrin (PE), anti-mouse IgG1-PE, anti-mouse IgG2a-PE, isotype control antibodies (mouse IgG1, mouse IgG2a), and antibodies against mouse CD16/32, CD3-PE-Cy7, CD45-APC-Cy7, B220-FITC, CD11b-APC, CD11c-BV421, CD11c-PE-Cy7, Siglec-F-FITC, Ly-6G-FITC, CD4-BV510, CD8α-Percp/Cy5.5, CCR3-Percp/Cy5.5, I-A/I-E-BV510, CD103-FITC and IL17A-APC were purchased from BioLegend (San Diego, CA, USA). Anti-mouse IL5-PE, anti-mouse IL-2-APC, and anti-mouse IL13-eFluor450 were purchased from Invitrogen (Thermo Fisher Scientific, Minato-ku, Tokyo, Japan). Mouse IL-17A and IL-2 recombinant proteins were purchased from BioLegend.

Murakami Y., Ishii T., Nunokawa H., Kurata K., Narita T, & Yamashita N. (2020). TLR9–IL-2 axis exacerbates allergic asthma by preventing IL-17A hyperproduction. Scientific Reports, 10, 18110.

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Multifaceted Immune Cell Characterization

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The single-cell suspensions were incubated for 3 h at 37°C with PMA (Sigma-Aldrich, p1585; 200 ng/ml), brefeldin A (BioLegend, 420601; 5 µg/ml), and ionomycin (Abcam, ab120116; 1 µg/ml). And then washed and stained with fixable viability stain 620 (FVS 620; BD Biosciences, 564996) for 10 min. Next, stain cells with following surface antibodies: γδT-PE-CY5 (15-5711-81, eBioscience). After performing surface staining as described above, 4% paraformaldehyde was used to fix cells and added PBS solution (containing 0.1% Triton X-100) to permeabilize the cell surface. Intracellular staining antibodies were included: IL17A-APC (506916, BioLegend). After staining for 30 min, the cells were washed by PBS and using the NovoCyte flow cytometer and ACEA NovoExpress™ software (ACEA Biosciences, San Diego, CA, USA) for analysis.

Zhao F., Zhang C., Li G., Zheng H., Gu L., Zhou H., Xiao Y., Wang Z., Yu J., Hu Y., Zeng F., Wang X., Zhao Q., Hu J., Yue C., Zhou P., Huang N., Hao Y., Wu W., Cui K., Li W, & Li J. (2022). A role for whey acidic protein four-disulfide-core 12 (WFDC12) in the pathogenesis and development of psoriasis disease. Frontiers in Immunology, 13, 873720.

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