The largest database of trusted experimental protocols

12 protocols using anti il 17a tc11 18h10.1

1

Comprehensive Immune Profiling by Flow Cytometry

Check if the same lab product or an alternative is used in the 5 most similar protocols
Fluorescence-conjugated anti-GM-CSF (MP1–22E9), anti-IL-10 (JES5), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD4 (RM4–5), anti-Thy1.1 (OX-7), anti-IFN-γ (XMG1.2), and anti-IL17A (TC11–18H10.1) from Biolegend, as well as fluorescence-conjugated Annexin V and 7-amino-actinomycin D from BD Biosciences were used. For intracellular cytokine staining, lymphocytes were stimulated for 4 hours with 50 ng/ml of PMA (phorbol 12-myristate 13-acetate) and 1 µM ionomycin in the presence of 5 µg /ml brefeldin A. Then 0.5–1×106 cells were fixed/permeabilized for 20 mins at 4 degree with Fixation/Permeabilization Solution Kit (BD Bioscience) and stained with fluorescently-conjugated antibodies per manufacture’s protocols. For surface staning, 0.5–1×106 cells were collected and stained with fluorescently-conjugated antibodies for 15 mins at 4 degree. After wash, stained cells were recorded on Canto (BD Biosciences) and analyzed by FlowJo software (Treestar).
+ Open protocol
+ Expand
2

Cytokine Production Quantification Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
Preparation and enzymatic digestion of tissues were as described elsewhere (11) (link). For assessment of IL-17A production, cells were stimulated for 5 h with 10 ng/ml of phorbol 12-myristate 13-acetate and 500 ng/ml of ionomycin (both Sigma-Aldrich, St. Louis, Missouri). The following antibodies were used: anti-mouse anti-CD11b (M1/70), anti-CD11c (N418), anti-CD19 (6D5), anti-CD31 (390), anti-CD45 (30-F11), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD115 (AFS98), anti-F4/80 (BM8), anti-Gr1 (RB6-8C5), anti-Il17ra (PAJ-17R), anti-MHCII (M5/114.15.2), anti–T-cell receptor β (H57-597), and anti–IL-17A (TC11–18H10.1) (BioLegend and eBioscience [San Diego, California]). Aqua and near-infrared LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen) was used according to the manufacturer’s instructions. Flow cytometry analysis was performed on a BD FACSCanto or LSR II (Becton-Dickinson, Franklin Lakes, New Jersey). Data were analyzed by using FlowJo software (Tree Star Inc., Ashland, Oregon). Gating was performed for live, CD45+ events.
+ Open protocol
+ Expand
3

Multiparametric Analysis of Immune Cell Cytokines

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were stimulated for 4 h with PMA (phorbol 12-myristate13-acetate; 50 ng/mL), ionomycin (1.0 µg/mL), and a protein-transport inhibitor containing monensin. PMA and ionomycin stimulate the immune cells in a non-antigen specific manner, and monensin is used to trap the cytokine within the cytosol. After stimulation, surface markers were stained for 15–20 min at room temperature in PBS with 1% FBS. Cells were then fixed in Cytofix and permeabilized with Perm/Wash Buffer using the BD Fixation Permeabilization solution kit and stained with anti-IL-17A (TC11-18H10.1, Biolegend, San Diego, CA, USA); anti-IFN-γ (XMG1.2, Biolegend); and anti-IL-4 (11B11, Biolegend) antibodies diluted in Perm/Wash buffer. Permeabilization was undertaken in order to make the intracellular cytokines accessible to the FACS antibodies. All antibodies were used in 1:500 dilutions. Flow cytometry was undertaken using BD FACS Symphony, and the data were analyzed with FlowJo software version 10.9.0.
+ Open protocol
+ Expand
4

Multiparametric Analysis of Murine Lymphocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols
Lymphocytes were isolated from various organs of 6–18 weeks old, age- and sex-matched mice of. Fluorescence-conjugated anti-CD4 (GK1.5), anti-CD25 (PC61.5), anti-CD62L (MEL-14), anti-CD44 (IM7), anti-CD45.1 (A20), anti-CD45.2 (104), anti-IFN-γ (XMG1.2), anti-Thy1.1 (OX-7) and anti-IL-17A (TC11-18H10.1) (Biolegend) were used. For intracellular cytokine staining, lymphocytes were stimulated for 4 hours with 50ng/ml of PMA (phorbol 12-myristate 13-acetate) and 1μM ionomycin in the presence of brefeldin A. Stained cells were analyzed on a FACSCanto (BD Biosciences) or were sorted on a MoFlo (DakoCytomation, Beckman Coulter). A figure exemplifying the gating strategy is provided in Supplementary Fig. 2.
+ Open protocol
+ Expand
5

CFSE-Labeled T Cell Activation Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols
Draining LN cells (axillary, brachial, and inguinal) were incubated in 0.2 mM CFSE for 8 min at room temperature (Life Technologies). CFSE-labeled cells were washed and cultured in medium only, with rhMOG, or with OVA protein (Sigma, St. Louis, MO) at 10 or 30 mg/ml for 96 h. Culture supernatants were collected for cytokine measurement, and cells were restimulated with 13 cell stimulation mixture (eBioscience) in the presence of 3 mg/ml brefeldin A (eBioscience) for 4 h. Surface Ags were stained with anti-TCRb, anti-CD4, and anti-CD19, and followed by the intracellular staining. For the detection of Foxp3 and intracellular cytokines, cells were treated with Foxp3 Fixation/Permeabilization buffer (eBioscience) and stained with anti-Foxp3 (NRRF-30; eBioscience) and anti–IL-17A (TC11-18H10.1; Biolegend) as an Ab pair or anti–IFN-g (XMG1.2; BD Biosciences) and anti–IL-17A as a pair in the FACS buffer.
+ Open protocol
+ Expand
6

Th17 Cell Induction from Naïve CD4+ T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
The polarized Th0 and Th17 cells from naïve CD4+ T cells were rinsed with PBS re-cultured in RPMI 1640 medium containing 2 mM glutamine and 10% FBS in the presence of 50 ng/mL phorbol 12-myristate 13-acetated (PMA) and 1 μM ionomycin for 6 hours. 1 μg/mL brefeldin A was added for the last 3 hours of culture (all purchased from Sigma Aldrich). Thereafter, cells were stained for surface markers (anti-CD4, GK1.5, BioLegend). Intracellular cytokine staining (anti-IL-17A, TC11–18H10.1, BioLegend) was performed as described previously (28 (link)). Approximately 100,000 cells were analyzed on a BD FACSCanto II flow cytometer (BD Biosciences). The Th17 population was defined as CD4+IL-17A+.
+ Open protocol
+ Expand
7

Multiparameter Flow Cytometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cell surface staining was performed using standard methods. The following antibodies were used: using anti-CD4 (GK1.5, Biolegend), anti-CD8 (53-6.7, BD Pharmingen San Jose, CA), anti-B220 (RA3-6B2, BD Pharmingen), anti-CD11b (M1/70, BD Pharmingen), and anti-CD11c HL3 (BD Pharmingen). Intracellular cytokine staining was performed on single cell suspensions prepared from lymph node and spleen. Cells were re-stimulated for 4 h in the presence of PMA (50 ng/mL)/ionomycin (1 mM, Sigma–Aldrich, St. Louis, MO), and monensin (eBioscience, San Diego, CA). Cells were then prepared using the intracellular fixation and permeabilization buffer set (eBioscience) according to the manufacturers instructions. Cells were stained with anti-IL17A (TC11-18H10.1 Biolegend, 17B7 eBioscience), or anti-IFN-γ (XMG1.2 Biolegend). All flow cytometry was performed on a FacsCanto II cytometer (BD Biosciences), and data were analyzed using FlowJo v X 10.0.7 software (Tree Star, Inc. Ashland, OR).
+ Open protocol
+ Expand
8

Multiparametric Analysis of Murine Lymphocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols
Lymphocytes were isolated from various organs of 6–18 weeks old, age- and sex-matched mice of. Fluorescence-conjugated anti-CD4 (GK1.5), anti-CD25 (PC61.5), anti-CD62L (MEL-14), anti-CD44 (IM7), anti-CD45.1 (A20), anti-CD45.2 (104), anti-IFN-γ (XMG1.2), anti-Thy1.1 (OX-7) and anti-IL-17A (TC11-18H10.1) (Biolegend) were used. For intracellular cytokine staining, lymphocytes were stimulated for 4 hours with 50ng/ml of PMA (phorbol 12-myristate 13-acetate) and 1μM ionomycin in the presence of brefeldin A. Stained cells were analyzed on a FACSCanto (BD Biosciences) or were sorted on a MoFlo (DakoCytomation, Beckman Coulter). A figure exemplifying the gating strategy is provided in Supplementary Fig. 2.
+ Open protocol
+ Expand
9

Murine Lung Cell Isolation and Characterization

Check if the same lab product or an alternative is used in the 5 most similar protocols
Murine lungs were chopped and incubated in RPMI1640 media with 1 mg/mL type IV collagenase (Worthington BioChem, Lakewood, NJ, USA) for at least 1 h 30 min at 37 °C. Red blood cells (RBC) were lysed using RBC lysis buffer (Biolegend, San Diego, CA, USA). Lung single cells were stained with following fluorochrome-conjugated antibodies. For surface staining, the following antibodies were used. Anti-CD45 (30-F11), Anti-CD3e (145-2C11), anti-CD11c (HL3), anti-CD11b (M1/70), anti-CD19 (ID3), anti-CD49b (DX5), anti-FcεRIα (MAR-1), anti-CD90,2 (30-H12), anti-F4/80 (BM8) and anti-Ly6G (1A8), purchased from Biolegend. Anti-SiglecF (E50-2440) was purchased from BD Bioscience (San Diego, CA, USA). For intracellular staining, the following antibodies were used: anti-IL5 (TRFK5) and anti-IL17A (TC11-18H10.1), purchased from Biolegend (San Diego, CA, USA). Anti-IL13 (eBio13A) was purchased from Thermo Fisher Scientific. For intracellular staining, a Fixation/Permeabilization Solution Kit with BD GolgiPlug (BD Biosciences, San Diego, CA, USA) was used following the manufacturer’s protocol. Flow cytometry was carried out using LSRFortessa™ X-20 (BD biosciences, San Jose, CA, USA) and analyzed by FlowJo (V10.2) software (BD biosciences, San Jose, CA, USA). The gating strategy for immune cell population was represented in Supplementary Figure S1.
+ Open protocol
+ Expand
10

Immune Cell Phenotyping Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
Isotype control Abs (IgG1, IgG2a, and IgG2b), CD1d (1B1), CD5 (53-7.3), CD19 (1D2/CD19), CD4 (GK1.5), CD25 (3C7), anti-FoxP3 (MF-14), anti-IL-10 (JES5-16E3), and anti-IL-17A (TC11-18H10.1) were purchased from BioLegend (San Diego, CA, USA).
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!