Draining lymph node cells were collected for further analysis at the end of CIA as described above. For regulatory T cell analysis, the cells were surface-stained with antibodies against CD4 and CD25, followed by intracellular staining with anti-Foxp3 antibody (eBioscience; San Diego, CA). For the Th1 and 17 cells analysis, the lymph node cells were stimulated with PMA (50 ng/ml; Sigma-Aldrich, St. Louis, MO, USA) plus ionomycin (1 μg/ml; Sigma-Aldrich, St. Louis, MO, USA) for 5 h in culture medium in the presence of GolgiStop (BD Biosciences, San Jose, CA, USA), then surface-stained with anti-CD4 antibody followed by intracellular staining with anti-IL-17 and anti-IFN-γ antibodies (eBioscience; San Diego, CA). The cells were analyzed on an LSRII flow analyzer (BD Biosciences, San Jose, CA, USA) using FlowJo (Tree Star, Ashland, OR) (Qiao et al. 2012 (link); Ying et al. 2010 (link);).
Anti il 17
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-IL-17 is a laboratory reagent used for the detection and quantification of the cytokine Interleukin-17 (IL-17) in biological samples. It functions as a specific antibody that binds to IL-17, allowing for its identification and measurement.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 4, by Thermo Fisher Scientific (7 mentions)
Anti ifn γ, by Thermo Fisher Scientific (5 mentions)
Ionomycin, by Merck Group (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Anti foxp3, by Thermo Fisher Scientific (3 mentions)
Anti ifn γ, by BD (3 mentions)
Anti ifn γ antibody, by Thermo Fisher Scientific (2 mentions)
Anti cd4, by BD (2 mentions)
Anti rorγt, by Thermo Fisher Scientific (2 mentions)
Anti il 22, by Thermo Fisher Scientific (2 mentions)
Golgiplug, by BD (2 mentions)
Anti cd4, by Thermo Fisher Scientific (2 mentions)
Anti il 10, by BioLegend (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Anti ifn γ, by BioLegend (2 mentions)
Anti pd 1, by Thermo Fisher Scientific (2 mentions)
Anti cd8, by BioLegend (2 mentions)
Anti il 17, by R&D Systems (2 mentions)
Multiscreen ha filter plate, by Merck Group (2 mentions)
Streptavidin alkaline phosphatase, by Vector Laboratories (2 mentions)
20 protocols using anti il 17
1
Regulatory T Cell and Th17 Analysis
2
Intracellular Cytokine Staining and Caspase-1 Activity Assay
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For intracellular staining, cells were incubated for 4–5 h with 25 ng/ml PMA
(Sigma-Aldrich), 2 mg/ml ionomycin (Sigma-Aldrich), and 10 mg/ml brefeldin A
(eBioscience) at 37°C/5% CO2. After surface staining with
fluorescent-labeled anti-CD4 (BD Bioscience), cells were re-suspended in
fixation/permeabilization buffer (eBioscience) for intracellular staining with
fluorescent-labeled anti-IL-17 (eBioscience). Caspase-1 activity was detected
with FLICA kit (Immunochemistry, Shanghai, China), according to the
manufacturer’s instructions. Flow cytometry was performed on a FACSCalibur
instrument (BD Biosciences) and analysis was done using FlowJo software (Tree
Star, Ashland, OR).
(Sigma-Aldrich), 2 mg/ml ionomycin (Sigma-Aldrich), and 10 mg/ml brefeldin A
(eBioscience) at 37°C/5% CO2. After surface staining with
fluorescent-labeled anti-CD4 (BD Bioscience), cells were re-suspended in
fixation/permeabilization buffer (eBioscience) for intracellular staining with
fluorescent-labeled anti-IL-17 (eBioscience). Caspase-1 activity was detected
with FLICA kit (Immunochemistry, Shanghai, China), according to the
manufacturer’s instructions. Flow cytometry was performed on a FACSCalibur
instrument (BD Biosciences) and analysis was done using FlowJo software (Tree
Star, Ashland, OR).
3
Multiparametric Flow Cytometry of T-Cells
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Five replicates of splenocytes (105) were plated in 200 µl of medium supplemented with 0.05 µg/ml PMA (Sigma-Aldrich), 0.001 µg/ml ionomycin calcium salt (Sigma-Aldrich) and 0.2 µl BD GolgiPlug (brefeldin A, BD Pharmingen). As control, the cells were stimulated with PMA and Ionomycin without GolgiPlug. After 4 h of incubation at 37°C and 5% CO2, 100 µl of the supernatant was frozen at −20°C for ELISA. The cells were first stained with anti-CD8α and anti-CD4 before following the instruction for cytokines’ intracellular staining with a mix of anti-IL-2, anti-IL-4, anti-IL-10, anti-IL-17, anti-IL-22 and anti-IFN-γ, or intracellular staining for transcription factors with a mix of anti-Foxp3, anti-T-bet, anti-ROR-γt and anti-Gata-3 (eBioscience). The staining of intracellular cytokines and enzymes of CD8+ T-cells was done with a mix of anti-IL-2, anti-IFN-γ, anti-granzyme B and anti-perforin (reagents and isotypes from eBioscience). For FACS analysis, cells were resuspended in 50 µl PBS (1x).
4
Intracellular Cytokine Analysis in BALF
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Broncheoalveolar lavage was performed with five 1.0-ml aliquots of PBS through a tracheal cannula. To evaluate cytokine production, BALF cells were harvested and restimulated with 50 ng/ml PMA and 1 µg/ml ionomycin (Sigma-Aldrich) for 4 h. For intracellular staining, brefeldin A (BD-Pharmingen, San Diego, CA) was added during the final 2 h of stimulation. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and incubated with anti-CD4, anti-γδTCR, anti-IL-17, and anti-IFN-γ antibodies (eBioscience). Intracellular cytokine levels were assayed by flow cytometry.
5
Multiparameter Flow Cytometric Profiling of Immune Cells
6
Intracellular Cytokine and Treg Staining
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For intracellular IFN-γ ⁄ IL-4 ⁄ IL-17 staining and detection, 2 × 106 splenocytes, lymphocytes, or liver cells from schistosome-infected or normal mice were surface stained with anti-CD3-APC mAbs (eBioscience, San Diego, CA) and anti-CD4-FITC mAbs for 30 minutes. Cells were washed, fixed and permeabilized with Cytofix/Cytoperm buffer (BD Pharmingen) for 40 minutes and then intracellularly stained with PE-conjugated anti-IFN-γ, anti-IL-4 or anti-IL-17 respectively (eBioscience) for 60 minutes. Cells were gated on the CD3+ population for analysis of Th1, Th2, or Th17 cells.
For detecting the proportion of CD4+ CD25+ Treg cells, intracellular Foxp3 staining was performed according to the manufacturer’s protocol of the Mouse Regulatory T Cell Staining Kit (eBioscience). Briefly, 2 × 106 splenocytes, lymphocytes or liver cells from schistosome-infected or normal mice were surface-stained with anti-CD3-PerCP, anti-CD4-FITC and anti-CD25-APC for 30 minutes, followed by fixation and permeabilization with Cytofix/Cytoperm buffer (BD PharMingen) for 40 minutes and intracellular staining with anti-Foxp3-PE for 15 minutes. Cells were gated on the CD3+CD4+ population for analysis of Treg cells.
For detecting the proportion of CD4+ CD25+ Treg cells, intracellular Foxp3 staining was performed according to the manufacturer’s protocol of the Mouse Regulatory T Cell Staining Kit (eBioscience). Briefly, 2 × 106 splenocytes, lymphocytes or liver cells from schistosome-infected or normal mice were surface-stained with anti-CD3-PerCP, anti-CD4-FITC and anti-CD25-APC for 30 minutes, followed by fixation and permeabilization with Cytofix/Cytoperm buffer (BD PharMingen) for 40 minutes and intracellular staining with anti-Foxp3-PE for 15 minutes. Cells were gated on the CD3+CD4+ population for analysis of Treg cells.
7
Intracellular Cytokine Profiling of T-cells
8
Lung and Tumor Immune Cell Isolation
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Lymphocytes were isolated from lung and tumor tissue by digestion with collagenase A (1 mg/ml; Roche) and DNase I (0.5 µg/ml; Roche) in isolation buffer (RPMI 1640 supplemented with 5% FBS, 1% l -glutamine, 1% penicillin-streptomycin, and 10 mM Hepes) for 30 min at 37°C. Cells were filtered through 100-µm cell strainers, washed in isolation buffer, and stained in PBS supplemented with 0.25% BSA, 2 mM EDTA, and 0.1% sodium azide. Antibodies used included anti-CD45, anti-Foxp3, anti–IL-18Rα, anti-ST2, anti-CD62L, anti-CD103, anti–PD-1, anti-GITR, anti–CTLA-4, anti-KLRG1, anti-Ki67, anti-CD5, anti-NK1.1, anti-CD45R (B220), anti–IL-17, anti–IL-4, anti-IFNγ, and anti–Ly-6C (eBioscience); anti-TCRβ, anti-CD3, anti-CD4, anti-CD8, anti-CD127, anti-CD11B, anti-MHCII, and anti-Gr1 (BioLegend); anti-CD44 and anti-CD25 (Tonbo); anti–IL-5 and anti-TNFα (BD PharMingen); and anti-AREG (R&D Systems). For exclusion of dead cells, samples were first stained with Ghost Dye (Tonbo) cell viability reagent. Intracellular staining for cytokines, Foxp3, AREG, Ki-67, and CTLA-4 was performed using the Foxp3/transcription factor staining buffer set (eBioscience) as per manufacturer’s protocol. The H2-Kb OVA257–264 tetramer was obtained from the NIH Tetramer Core Facility.
9
Collagen-Induced Arthritis Mouse Model
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Female DBA/1 mice of 6 to 8 weeks old were provided by the Center for Animal Experiment and ABSL-3 Laboratory, Wuhan University School of Medicine, Hubei, China. The following reagents were used in this study: EP4 receptor antagonist L161982 (N-[[4′-[[3-Butyl-1,5-dihydro-5-oxo-1-[2-(trifluoromethyl)phenyl]-4H-1,2,4-triazol-4-yl]methyl] [1,1′-biphenyl]-2-yl]sulfonyl]-3-methyl-2-thiophenecarboxamide) (Tocris, UK); Chicken Type II Collagen (Sigma, USA); Celecoxib, PGE2 and BrdU Cell Proliferation ELISA kit (Abcam, USA); Dimethyl sulfoxide (DMSO, Sigma, USA); Freund’s Complete Adjuvant (Chondrex, USA); Interleukin-17 (IL-17), monocyte chemoattractant protein-1 (MCP-1), and ELISA kit (eBioscience, USA); Mouse antibodies: FITC-anti-CD4, PE-anti-CD25, PeCY5-anti-Foxp3, PE-CD62L and PeCY5-anti-IL-17, anti-IL-17, anti-MCP-1, anti-cleaved-caspas 3, soluble anti-CD3 and soluble anti-CD28 (eBioscience, USA); EasySep mouse CD4+CD62L+ naïve T cells isolation kit (Milenyi biotec, USA); Th17 cells inducement: mouse TGF-β1, IL-6, IL-23, anti-IFNγ and anti-IL-4 (Milenyi biotec, USA).
10
ELISpot Assay for Antigen-Specific T-cells
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Lung suspensions from Mtb-infected mice were prepared as described previously11 (link) and were used in ELISpot assays as described below. Antigen-specific IFN-γ-producing and IL-17-producing cells were analyzed by ELISpot assay. Multi-screen HA filter plates (Millipore, Billerica, MA) were coated with anti-IFN-γ (BD Biosciences) or anti-IL-17 (R&D Systems) antibodies. Single cell suspensions were added to the plate at a starting concentration of 1×105 cells/well and doubling dilutions made. Cells were cultured overnight in the presence of 1×106 irradiated splenocytes and 10 μg/mL ESAT-61-20 peptide and 10 U/mL recombinant mouse IL-2. The following day, biotinylated anti-IFN-γ or anti-IL-17 antibody (both from eBioscience, San Diego, CA) was added and incubated overnight. Plates were developed by incubation with streptavidin-alkaline phosphatase (Vector Labs, Burlingame, CA) for two hours, followed by incubation with NBT/BCIP (Sigma Aldrich). Spots were enumerated using a CTL-ImmunoSpot analyzer (CTL, Shaker Heights, OH) and the frequency and total number of responding cells calculated as described before11 (link).
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