Splenic IFNγ, IL-5, or IL-17 cytokine-secreting cells were detected by FluoroSpot assay. Briefly, the membrane of 96-well IPFL-bottomed microplates (Millipore) were coated with rat anti-mouse IFNγ, IL-5, or IL-17 antibodies (PharMingen) at 10 μg/mL, incubated overnight at +4°C, washed and blocked with RPMI containing penicillin-streptomycin, L-Glutamine and β-mercaptoethanol (all from Gibco) and 10% FCS (Hyclone). 1 × 106 freshly isolated splenocytes/well were incubated with a mix of PT (2.5 μg/mL), PRN (5 μg/mL), FIM2,3 (5 μg/mL), and FHA (5 μg/mL) antigens in presence of 10 U/mL of murine IL-2 (Bohringer Mannhein). After 24 (IFNγ and IL-17) or 48 (IL-5) h, biotinylated anti-mouse IFNγ (2 μg/mL), IL-5 (1 μg/mL), or IL-17 (1 μg/mL) antibodies (PharMingen) were added for 2 h at RT. Streptavidin-PE (Southern Biotech) at 1 μg/mL was added for 1 h at RT. The plates were stored at +5°C ± 3°C in the dark until reading. Each spot, corresponding to one IFNγ-, IL-5-, or IL-17-secreting cell, was enumerated with an automatic ELISPOT fluorescent plate reader (Microvision). Results were expressed as number of IFNγ-, IL-5-, or IL-17-secreting cells per 1 × 106 splenocytes.
Il 17
Manufactured by BD
Sourced in United States
IL-17 is a cytokine that plays a crucial role in the immune system. It is involved in the regulation of inflammatory responses and the recruitment of neutrophils to sites of inflammation. IL-17 is produced by various immune cells, including T-helper 17 (Th17) cells, and is important in host defense against extracellular pathogens.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ, by BD (29 mentions)
Interleukin 4 (il 4), by BD (11 mentions)
Tnf α, by BD (11 mentions)
Il 10, by BD (8 mentions)
Interleukin 2 (il 2), by BD (6 mentions)
Interleukin 6 (il 6), by BD (6 mentions)
Ionomycin, by Merck Group (5 mentions)
Cd62l, by BD (4 mentions)
T bet, by BD (4 mentions)
Cd11b, by BD (4 mentions)
Ifn γ, by Thermo Fisher Scientific (4 mentions)
Golgiplug, by BD (4 mentions)
Il 13, by BD (3 mentions)
Il 22, by BD (3 mentions)
Golgistop, by BD (3 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (3 mentions)
Rorγt, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Brefeldin a, by Merck Group (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
49 protocols using il 17
1
Cytokine-Secreting Cell Detection in Spleen
2
Multiparametric Flow Cytometry Immune Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells suspensions were prepared from lymph nodes and pancreatic islets. Single-cell suspensions were labeled with fluorochrome-conjugated monoclonal antibodies: anti-mouse CD3, CD4, CD8, ST2, and CXCR3 (BD Biosciences), CD11c and CD11b antibodies (BioLegend, San Diego, CA) or with isotype-matched control and analyzed on a FACSCalibur (BD) using CELLQUEST software (BD). The intracellular staining was performed with lymph node cells incubated for 6 h in the presence of Phorbol 12-myristate13-acetate (50 ng/ml) (Sigma, USA), Ionomycin (Sigma, USA) (500 ng/ml), and GolgyStop (BD Pharmingen) at 37°C, 5% CO2, stained with anti-CD4 monoclonal antibodies or appropriate isotype controls, fixed and permeabilized with a Cytofix/Cytoperm solution. Intracellular staining was performed using monoclonal antibodies: IFN-γ, IL-17, IL-10, IL-5, IL-13, IL-2, and Foxp3 (BD Biosciences) or appropriate negative controls. Cells were analyzed with the FACSCalibur Flow Cytometer (BD Biosciences), and analysis was conducted with FlowJo (Tree Star).
3
T Cell Subpopulation Immunophenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were stained with fluorescence-conjugated antibodies for markers of Tregs and Th cells, including CD4, IFN-γ, IL-9, IL-17, IL-22, and Foxp3 (BD Pharmingen), according to the manufacturer’s protocols. For cytokine staining, the cells were stimulated using a Leukocyte Activation Cocktail with BD GolgiPlug™ for 6 h at 37 °C with 5% CO2. For intranuclear staining, the cells were mixed with the fixation/permeabilization working solution and incubated for 30 min. Flow cytometric analysis of antibody-labeled cells was performed on a BD Accuri C6 system. The details were provided in the supplementary material . The numbers of cell subpopulations were calculated by multiplying the total cell number by the percentages of each subpopulation, as determined by flow cytometry.
4
Dendritic Cell Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant GM-CSF, IL-4, and naïve CD4 T cell enrichment kit, Abs to IL-12p40/70, TNF-α, IL-6, IFN-γ, IL-17, PE-Cy7-CD11c, FITC-CD80, PE-CD86, efluor-MHCII, APC-CD40, STAT-1, pSTAT-1, STAT-4, pSTAT-4, JNK, and pJNK were purchased from BD Biosciences (San Diego, CA, USA). Biotinylated anti-CD83 and APC-conjugated CCR7 Abs were purchased from eBioscience (San Diego, CA, USA). Abs against actin were from Sigma (St. Louis, MO, USA), SOCS-3 from Abcam (Cambridge, MA, USA) and anti-pERK and ERK Abs from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The endotoxin removal column was purchased from Thermo Scientific (Rockford, IL, USA).
5
Intracellular Cytokine Profiling by Flow Cytometry
6
Treg Cell Differentiation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total CD4+ T cells were isolated from lymph nodes of FoxP3 EGFP mice and cultured ex vivo in media supplemented with 20 nM retinoic acid (sigma), 5 or 10 ng/mL TGFβ (R&D systems) and recombinant IL-2 (R&D systems) at either 100 or 250U/mL. When assessing the effects of okadaic acid on Treg cell differentiation, the lower concentrations of TGFβ and IL-2 were used. The higher concentrations of TGFβ and IL-2 were used to generate maximum numbers of Tregs. Cells were plated on dishes coated with anti-CD3 and anti-CD28 (clones 17A2 and 37.51 respectively; eBiosciences). Cultures were incubated for 72 hours and flow cytometry was used to assess FoxP3 induction and CD25 expression. To assess cytokine production, GolgiPlug (BD Biosciences) was added to the culture for 5 hours prior to staining for IFNγ (clone XMG1.2; eBioscience) and IL-17 staining (clone TC11-18H10; BD).
7
Multiparametric Flow Cytometry Analysis
8
Western Blot Analysis of AMPK and Foxp3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells or spleen tissues were homogenized in RIPA lysis buffer (Santa Cruz), and protein contents were measured using the Bradford (BCA) assay (Pierce). Anti-phospho-AMPK (CST), AMPK (CST), Foxp3 (CST), IL-17 (BD), and Gapdh (Santa Cruz) was used. Samples were subjected to Western blot as described previously [29 (link)].
9
Immunophenotyping of Renal Transplant Patients
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The peripheral blood of control, chronic renal allograft dysfunction (CRAD) with AMR, CRAD without AMR patients after renal transplantation was collected, mononuclear cells were separated by the lymphocytes separation medium (TBD), then labeled by the flow type antibodies CD4, CXCR5, Foxp3, ICOS, IL-17, IL-4, IFN-γ, CD127, CXCR3 and CCR6 (All purchased from BD), and sent to the flow cytometry after three washes using PBS.
Renal biopsies of all groups of patients as mentioned above were selected and cut into slices in 4 µm thickness after the formalin fixation and paraffin embedding. The primary antibodies CD4, CXCR5 and Foxp3 (All purchased from Abcam), were added and stay overnight at 4 °C. After three washes using PBS, fluorescent secondary antibody was added and incubated in 37 °C thermostat for 30 min, and observed under the fluorescent microscope after three washes using PBS. We randomly selected 7 horizons in each biopsy and counted positive cell number.
Renal biopsies of all groups of patients as mentioned above were selected and cut into slices in 4 µm thickness after the formalin fixation and paraffin embedding. The primary antibodies CD4, CXCR5 and Foxp3 (All purchased from Abcam), were added and stay overnight at 4 °C. After three washes using PBS, fluorescent secondary antibody was added and incubated in 37 °C thermostat for 30 min, and observed under the fluorescent microscope after three washes using PBS. We randomly selected 7 horizons in each biopsy and counted positive cell number.
10
Cytokine Detection in Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytokines were detected in culture supernatants using capture ELISA. IFN-γ, TNF-α, IL-10, IL-6, IL-17, and IL-12p70, all purchased from BD PharMingen (United States), were used as paired monoclonal antibodies in combination with recombinant cytokine standards. Assays were performed according to the manufacturer’s guidelines.
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!