Single cell suspensions of spleen or draining lymph node were immunostained using various combinations of the following fluorescence-conjugated antibodies: CD4 (RM4-5; eBioscience), CD25 (37.51; BioLegend), Foxp3 (FJK-16 s; eBioscience), IL-17 (eBio17B7, eBioscience), TNF-α (MP6-XT22; BD Pharmingen), IFN-γ (XMG1.2; eBioscience), IL-4 (11B11; BD Pharmingen), and IL-10 (JES5-16E3; eBioscience). These cells were also intracellularly stained with the following antibodies: TNF-α, IL-17, IFN-γ, IL-10, and Foxp3. Prior to intracellular staining, cells were restimulated for 4 h with 25 ng/ml phorbol myristate acetate (PMA) (Sigma-Aldrich) and 250 ng/ml ionomycin (Sigma-Aldrich) in the presence of GolgiSTOP (BD Pharmingen). Intracellular staining was conducted using an intracellular staining kit (eBioscience) according to the manufacturer’s protocol. Flow cytometric analysis was performed on a FACS_LSR Fortessa (BD Pharmingen).
Il 17 ebio17b7
Manufactured by Thermo Fisher Scientific
The IL-17 (eBio17B7) is a lab equipment product from Thermo Fisher Scientific. It is a reagent that can be used to detect and quantify the presence of the interleukin-17 (IL-17) cytokine in biological samples. The core function of this product is to facilitate the analysis and measurement of IL-17 levels, which is important for various research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ xmg1.2, by Thermo Fisher Scientific (3 mentions)
Ionomycin, by Merck Group (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Golgistop, by BD (1 mentions)
Foxp3 fjk 16s, by Thermo Fisher Scientific (1 mentions)
Cd4 rm4 5, by Thermo Fisher Scientific (1 mentions)
Intracellular staining kit, by Thermo Fisher Scientific (1 mentions)
Il 10 jes5 16e3, by Thermo Fisher Scientific (1 mentions)
Tnfα mp6 xt22, by BD (1 mentions)
Il 4 11b11, by BD (1 mentions)
Facs lsr 2, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Fix perm buffer, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Cd3 17a2, by BioLegend (1 mentions)
P38 inhibitor sb203580, by Cell Signaling Technology (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
6 protocols using il 17 ebio17b7
1
Multiparametric Flow Cytometry Analysis
2
Immunophenotyping Murine Central Nervous System
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were euthanized two weeks post EAE induction. They were perfused with 30cc of PBS, and brains, spinal cords, and spleens were isolated. A single cell suspension was generated from each organ. Immune cells from the CNS were isolated using a 30% Percoll gradient. For intracellular staining, CNS mononuclear cells, were stimulated for 4 hours in DMEM (Gibco) containing 10% FBS (Gibco), GolgiPlug (BD Biosciences), 10ng/ml PMA, and 500ng/ml ionomycin. Antibodies for the cell surface markers were added to the cells in PBS with 2% FBS for 30 min on ice. After washing, cells were resuspended in Fix/Perm buffer (eBiosciences) for 30-45 minutes on ice, washed twice, and incubated with Abs for intracellular antigens (cytokines and transcription factors) in Perm buffer (30 min, on ice). Fluorescently conjugated antibodies directed against CD4 (clone RM4-5), CD8 (clone 53-6.7), CD3 (clone eBio500A2), IFN-γ (clone XMG1.2), Foxp3 (clone FJK-16s), and IL-17 (eBio17B7) were all purchased from eBiosciences. Samples were acquired using a FACS LSR II (BD Biosciences) and analyzed with FlowJo software (Tree Star, Inc.).
3
Multicolor Flow Cytometry for Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescein isothiocyanate-, R-phycoerythrin (PE)-, peridinin chlorophyll protein (PerCP)-, PE-Cy5-, PE-Cy7-, allophycocyanin (APC)-conjugated mAbs in this study include those to CD3 (17A2), CD4 (RM4-5), CD8 (53-6.7), F4/80 (BM8), CD11c (N418) from Biolegend, IFN-γ (XMG1.2 ), and IL-17 (eBio17B7 ) from eBioscience. For intracellular staining, cells were fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences). Dead cells were detected by LIVE/DEAD Fixable Green Dead Cell Kit (Invitrogen-Molecular Probes). Multiparameter flow cytometric analysis was performed on the FACS Verse (BD Biosciences, San Jose, Calif) and CytoFLEX (Beckman Coulter, China). Data were analyzed using FlowJo software (Ver. 8.8.7).
4
Investigating NFAT Signaling in T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse-specific antibodies against p38α (5F11; Cell Signaling Technology), CD4 (RM4-5; BD Biosciences), CD8 (53–6.7; BD), IL-17 (eBio17B7; eBioscience), and IFN-γ (XMG1.2; eBioscience) were used. Anti-CD3 (145-2C11) and anti-CD28 (37.51) were purchased from BD. Phorbol myristate acetate (PMA) and ionomycin were purchased from Sigma-Aldrich. Recombinant NFATc1 protein was purchased from SignalChem and anti-NFATc1 antibody (MA3-024) was purchased from Thermo Fisher Scientific. Anti–human phospho NFATc1 (S172; clone 679340) was purchased from R&D Systems. Anti-IRF4 antibody was purchased from Cell Signaling Technology, and IRF8, from Invitrogen. Recombinant full-length NFATc1 was purchased from SignalChem. SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) were purchased from Cell Signaling Technology. [γ-32P]ATP was purchased from PerkinElmer.
5
Immunofluorescence Analysis of Neutrophil Markers
6
Isolation and Analysis of Intestinal Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleens and mesenteric lymph nodes (mLNs) were removed from mice and disaggregated through a 100 μm sieve. Small intestines were excised and lamina propria lymphocytes (SILP) were prepared essentially as described [80 (link)] with slight modification in the tissue digestion step (digestion medium used was RPMI with 10% Foetal calf serum, 0.1% w/v collagenase type I and Dispase II (both Invitrogen), and tissue was digested for 30 min at 37°C). Cell suspensions were blocked with anti-FcγR antibody (clone 24G2; eBioscience) before labelling with antibodies specific for CD3 (eBio500A2), CD4 (clone GK1.5; eBioscience), Foxp3 (clone FJK-16s; eBioscience), IL-13 (clone eBiol13A; eBioscience), IFNγ (clone XMG1.2; eBioscience), IL-17(eBio17B7; eBioscience), IL-9 (RM9A4e; Biolegend) or p-Smad 2/3 (Santa Cruz). For intracellular cytokine analysis cells were incubated for 12 hours with 1x Cell stimulation cocktail (plus protein inhibitors) (ebioscience). Cells were then stained with antibodies using the eBioscience Foxp3 permibilization kit according to the manufacturer's instructions. For pSmad2/3 staining, an Alexa Fluor 594-labelled donkey anti-goat secondary antibody was used (Invitrogen). All samples were analysed on a FACS LSRII.
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
Revolutionizing how scientists
search and build protocols!