Human CD3 (BD Bioscience), CD4 (BD Bioscience), TCRgd (BD Bioscience), TCR Vd1 (Invitrogen), TCR Vδ2 (Invitrogen), IL-17A (BD Bioscience), and IFN-γ (BD Bioscience) mAbs were purchased from BD bioscience or Thermo Fisher Scientific. In brief, for cell surface staining, 0.5 × 105 cells/100 μl staining buffers were incubated with a cocktail for 30 min at 4°C. For intracellular staining, cells were fixed and permeabilized (BD Bioscience) and then stained intracellularly for IL-17A and IFN-γ. Flow cytometry analysis was performed on BD FACS Calibur (BD Bioscience) and analyzed with FlowJo software (Flowjo LLC, Ashland, OR).
Il 17a
Manufactured by BD
Sourced in United States, Israel
IL-17A is a cytokine that plays a role in the inflammatory response. It is involved in the recruitment and activation of neutrophils. IL-17A is a useful target for research related to inflammatory and autoimmune diseases.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ, by BD (21 mentions)
Cd11b, by BD (9 mentions)
Interleukin 4 (il 4), by BD (8 mentions)
Ionomycin, by Merck Group (8 mentions)
Phorbol myristate acetate (pma), by Merck Group (7 mentions)
Ifn γ, by Thermo Fisher Scientific (6 mentions)
Tnf α, by BD (6 mentions)
Cytofix cytoperm, by BD (5 mentions)
Ionomycin, by BD (5 mentions)
Golgiplug, by BD (5 mentions)
Il 10, by BD (5 mentions)
Fix perm, by BD (5 mentions)
Interleukin 6 (il 6), by BD (4 mentions)
Golgi stop reagent, by BD (4 mentions)
Cd103, by BD (4 mentions)
Dnase 1, by Merck Group (4 mentions)
Siglec f, by BD (4 mentions)
Interleukin 2 (il 2), by BD (4 mentions)
Lsrfortessa, by BD (4 mentions)
Anti cd45, by BD (3 mentions)
54 protocols using il 17a
1
Immunophenotyping of Human T Cells
2
Th17 and Treg Cell Detection Protocol
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The spleen tissue was aseptically removed and prepared into a single-cell suspension. For Th17 (CD3+CD4+IL17A+) cells and Treg cells (CD3+CD4+ CD25 + FOXP3+) detection, the cells were stimulated by cocktail A for 4 h (BD, Cat# 550583). Samples were then washed and re-suspended in 1 × PBS stained with FVS 780 (BD, Cat#565388) to discriminate viable cells and then incubated with various surface markers. Consequently, samples were fixed by eBioscience Fix/Perm (Cat#00-5523-00) or BD Fix/Perm buffer kit (Cat#554714) to destroy the cell membrane and then were stained with FOXP3 (eBioscience, Cat#17-5773-82), IL-17A (BD, Cat# 564169). In the study, CD3 (BD, Cat# 557,666), CD4 (BD, Cat# 552,775) and CD25 (BD, Cat# 558642) were used. Finally, samples were analyzed with the LSR Fortessa cell analyzer (BD) and BD FACSDiva 8.0.3 software.
3
Comprehensive Immunological Assays for Neuroinflammatory Disorders
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Fluoxetine hydrochloride (Tocris Bioscience, USA) was reconstituted in phosphate buffered saline (PBS). Digoxin (Sigma, USA) was dissolved in dimethyl sulfoxide and further diluted appropriately in PBS to get the desired concentration at the time of injection. Peptides viz. MOG(35-55), PLP(131-151) acquired from Anaspec, USA used for disease induction were dissolved in PBS and emulsified in Freund's complete adjuvant (Sigma, USA). Antibodies anti-CD3, anti-CD4, PE-anti-TCRβ, APC-anti-IFNγ, FITC-anti-IL-17A, anti-IL-4, anti-IL-23, Alexa Fluor 488-anti-rat, Alexa Fluor 555-anti-rabbit, cytokines, IL-12, IL-17A, IL-21, IL-23, TGFβ, IL-6 were purchased from BD Pharmingen, Abcam, Cell Signaling Technology, and RnD Technologies. Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (certified) were procured from Gibco, USA.
4
Comprehensive Treg Immunophenotyping and Epigenetics
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In vitro-expanded Tregs were stained with Live/Dead Aqua (Life Technologies) followed by the following anti-human antibodies; CD4 BUV496 (clone SK3, BD), CD127 APC-Vio770 (clone REA614, Miltenyi), CD25 APC (clone BC96, Biolegend) and TCR Vbeta21.3 PE (clone REA894, Miltenyi).
For intracellular staining: Tregs were stimulated with PMA/ionomycin and Brefeldin A (Sigma) for 4 h 37 °C after which cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and stained with anti-human antibodies; Foxp3 (clone 236A/E7, BD), Helios (clone 22F6, Biolegend), IFN-γ (clone B27, BD) and IL-17A (clone N49-653, BD). Cells were acquired on a Cytek Aurora 5 L flow cytometer and analysis performed in FlowJo (version 10.6.2). A gating strategy is found in Supplementary Fig.7 . Methylation of the FOXP3 TDSR was performed by pyrosequencing of intron 1 of TDSR region -2330 to -2263 from ATG of FOXP3 and analysis of 9 CpG sites by EpigenDx.
For intracellular staining: Tregs were stimulated with PMA/ionomycin and Brefeldin A (Sigma) for 4 h 37 °C after which cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and stained with anti-human antibodies; Foxp3 (clone 236A/E7, BD), Helios (clone 22F6, Biolegend), IFN-γ (clone B27, BD) and IL-17A (clone N49-653, BD). Cells were acquired on a Cytek Aurora 5 L flow cytometer and analysis performed in FlowJo (version 10.6.2). A gating strategy is found in Supplementary Fig.
5
Multiparametric Flow Cytometry Profiling
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Flow cytometry was conducted on a BD LSRFortessa (BD Biosciences) and the data were further analyzed using FlowJo software (Treestar Inc.). Cells were stimulated in RPMI 1640 with 10% FBS (Lonza) and 2 μL of leukocyte activation cocktail With GolgiPlug (BD Bioscience) for 6 h at 37°C. The live cells were discriminated by Fixable Viability Stain 780 (Biolegend). Cell surfaces were stained at 4°C for 30 min with appropriate fluorescence-labeled antibodies against CD3, CD4, and TCRβ (BD Biosciences). After the fixation and permeabilization using cytofix-cytoperm (BD Biosciences), intracellular cytokines were stained using appropriate fluorescence-labeled antibodies to interferon γ (IFN-γ) and interleukin-17A (IL-17A) (BD Biosciences) for 30 min at 4°C.
6
Multiparametric Flow Cytometry Profiling
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Flow cytometry was conducted on a BD LSRFortessa (BD Biosciences) and the data were further analyzed using FlowJo software (Treestar Inc.). Cells were stimulated in RPMI 1640 with 10% FBS (Lonza) and 2 μL of leukocyte activation cocktail With GolgiPlug (BD Bioscience) for 6 h at 37°C. The live cells were discriminated by Fixable Viability Stain 780 (Biolegend). Cell surfaces were stained at 4°C for 30 min with appropriate fluorescence-labeled antibodies against CD3, CD4, and TCRβ (BD Biosciences). After the fixation and permeabilization using cytofix-cytoperm (BD Biosciences), intracellular cytokines were stained using appropriate fluorescence-labeled antibodies to interferon γ (IFN-γ) and interleukin-17A (IL-17A) (BD Biosciences) for 30 min at 4°C.
7
Intracellular Cytokine Profiling of T Cell Subsets
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After single cell preparation from the tumor cells as well as control kidneys as described above, CD4 + T cells isolated and were differentiated into Th1, Th2, and Th17 lineages. For intracellular cytokine analysis, cells were restimulated with 500 ng/ml of ionomycin and 50 ng/ml of PMA in the presence of Golgi Stop (BD Pharmingen) for 5 hr. Cells were then permeabilized with Cytofix/Cytoperm Kit (BD PharMingen) and analyzed for the expression of IL-4,IL-5,IL-9,IL-13, IL-17A or IFN-g (BD Pharming) by FACSCalibur analysis.
8
Isolation and Analysis of Murine Lung Cells
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Collagenase digestion of mouse lung tissue was previously described in 30 (link). In short, mice were euthanized by CO2 asphyxiation after which perfusion was carried out by injecting 3–5 mL of phosphate buffered saline (PBS) through the right ventricle of the heart. Lungs were resected, minced with scissors, and digested in complete DMEM containing 15 mg/mL collagenase A (Roche, Indianapolis, IN), and 2500 units of DNase I (Sigma-Aldrich, St. Louis, MO) for 30 m at 37°C. Digested tissue was disrupted through a 10 mL syringe, filtered through a 100 μM pore size nytex screen and centrifuged through a 20 % Percoll solution in serum free media. 10x106 cells were then stained with appropriately diluted fluorophore-conjugated antibodies for 30 m. For intracellular cytokine staining (ICS) cells were diluted to 1x106/mL and stimulated for 4 h with PMA (10 ng / mL), ionomycin (10 μM), and Golgi-stop reagent (BD Bioscience, San Jose, CA). Antibodies used in this study are as follows: anti-CD45, CD11b, CD4, IL-17A, MHCII (I-Ab), CD103, SiglecF (BD Bioscience, San Jose, CA), CD11c, IFNγ (eBioscience San Diego, CA), DLL1, DLL4, Jagged1, Jagged2, and CD64 (Biolegend, San Diego, CA).
9
Isolation and Analysis of Murine Lung Cells
10
Cytokine Profiling of Activated Splenocytes
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For intracellular cytokine staining, we stimulated splenocytes for 4 h with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml, Sigma) and ionomycin (200 ng/ml, Sigma) in the presence of Golgi Plug (BD Biosciences). We first surface stained the resulting cells with antibodies to mouse CD4 (BD Biosciences) and then stained them with antibodies to mouse IFN-γ (BD Biosciences) and IL-17A (BD Biosciences) or IL-4 (BD Bioscience) using a Cytofix/Cytoperm fixation/permeabilization kit (BD Biosciences) according to the manufacturer’s instructions.
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