Ab-stained cells were analyzed using FACScaliber (BD Biosciences, USA) CellQuest Software. Anti-CD4-PE, anti-CD4-APC, anti-CD8-FITC, anti-CD25-FITC, anti-CD44-PE, anti-CD62L-APC, anti-CD69-FITC, anti-CCR5-PE, anti-CXCR3-PE, and anti-CXCR4-PE, anti-IFNγ-BV421, anti-IL17A-PE, anti-Thy1.2-BV605 were purchased from BD Biosciences.
Anti il 17a pe
Manufactured by BD
Sourced in United States
Anti-IL-17A-PE is a fluorescently labeled antibody that binds to Interleukin-17A (IL-17A), a pro-inflammatory cytokine involved in various immune processes. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd4 fitc, by BD (10 mentions)
Anti foxp3 pe, by BD (7 mentions)
Ionomycin, by Merck Group (6 mentions)
Anti cd25 apc, by BD (4 mentions)
Anti il 4 pe, by BD (3 mentions)
Anti ifn γ apc, by BD (3 mentions)
Anti cd44 pe, by BD (3 mentions)
Anti cd62l apc, by BD (2 mentions)
Golgiplug, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Anti il22 apc, by R&D Systems (2 mentions)
Golgiplug, by Merck Group (2 mentions)
Facscanto 2 system, by BD (2 mentions)
Anti ifn γ pe, by BD (2 mentions)
Anti cd4 pe, by BD (2 mentions)
Anti cd8 pe cy7, by BD (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Anti ifn γ fitc, by BD (2 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
25 protocols using anti il 17a pe
1
Comprehensive Immune Cell Profiling
2
Analyzing T Cell Cytokine Profiles
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Lung cells were harvested at day 4 post-infection. Cells (0.5 x 106 cells/ml) were stimulated for 4 to 6 hours with anti-CD3 (clone 145-2C11; 0.1μg/ml) and anti-CD28 (clone 37.51; 1μg/ml) in the presence of Golgi-Stop (BD Biosciences). Stimulation with fungal ligands yielded comparable cytokine production by transgenic T-cells compared to CD3/CD28 stimulation as shown previously [42 (link),60 (link)]. After cells were washed and stained for surface CD4 and CD8 using anti-CD4 PerCP, anti-CD8 PeCy7, and anti-CD44-FITC mAbs (Pharmingen), they were fixed and permeabilized in Cytofix/Cytoperm at 4°C overnight. Permeabilized cells were stained with anti-IL-17A PE and anti-IFN-γ Alexa 700 (clone XMG1.2) conjugated mAbs (Pharmingen) in FACS buffer for 30 min at 4°C, washed, and analyzed by FACS. Cells were gated on CD4 and cytokine expression in each gate analyzed. The number of cytokine positive CD4+ T cells per organ was calculated by multiplying the percent of cytokine-producing cells by the number of CD4+ cells. Intracellular Caspase 3 Alexa647 (9602S, Cell Signaling), Caspase 8 PE (12602S Cell Signaling), Bcl2 Alexa 647 (633510 Biolegend) and Bcl-xL PE (13835 Cell Signaling) were stained from ex vivo derived lymph node cells and splenocytes.
3
Multiparametric Profiling of T Cell Subsets
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For the analysis of Th1, Th2, and Th17 cells, cells in 1 ml of heparin-anticoagulated venous blood were stimulated for 5 h with 10 μl of PMA, 10 μl of ionomycin (final concentration was 750 ng/ml), and 1 μl of GolgiStop, respectively, in a 37°C incubator. Stimulated cells (80 μl) were taken for surface staining, and the cells were fixed and permeabilized using fixation/permeabilization reagent (BD, USA) and then stained with anti-IFN-γ-APC (for Th1), anti-IL-4-PE (for Th2), and anti-IL-17A-PE (for Th17) monoclonal (Supplemental Table 2
For the analysis of CD4 Tregs, cells in 80 μl of heparin-anticoagulated venous blood were aliquoted into tubes without PMA and ionomycin stimulation and surface-labeled with anti-CD4-FITC and anti-CD25-APC followed by fixation, permeabilization, and intracellular staining with anti-FoxP3-PE (all from BD, USA). The labeled cells were washed and analyzed with a FACSCalibur flow cytometer (Becton-Dickinson) using the CellQuest software (BD, USA). At least 5,000 to 10,000 cells were collected to calculate the percentage of these CD4+ T subsets, and the total number of CD4+ T cells was calculated using the internal microsphere counting standard.
In this study, we used an equation to calculate the absolute number of CD4+ T subsets: the absolute number of CD4+ T subsets = the percentage of each CD4+ T subset * the absolute number of total CD4+ T cells.
For the analysis of CD4 Tregs, cells in 80 μl of heparin-anticoagulated venous blood were aliquoted into tubes without PMA and ionomycin stimulation and surface-labeled with anti-CD4-FITC and anti-CD25-APC followed by fixation, permeabilization, and intracellular staining with anti-FoxP3-PE (all from BD, USA). The labeled cells were washed and analyzed with a FACSCalibur flow cytometer (Becton-Dickinson) using the CellQuest software (BD, USA). At least 5,000 to 10,000 cells were collected to calculate the percentage of these CD4+ T subsets, and the total number of CD4+ T cells was calculated using the internal microsphere counting standard.
In this study, we used an equation to calculate the absolute number of CD4+ T subsets: the absolute number of CD4+ T subsets = the percentage of each CD4+ T subset * the absolute number of total CD4+ T cells.
4
Quantification of Cytokine Levels and T-Cell Subsets
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Antibodies against IL-6, p53 and IL-17R were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human recombinant IL-6 (Cat. no. 206-IL) and IL-17A (Cat. no. 7955-IL), and human neutralizing antibodies to IL-6 (aIL-6) (Cat. no. AF-206-NA) and IL-17A (aIL-17A) (Cat. no. AF-317-NA) were obtained from R&D Systems (Minneapolis, MN, USA). Enzyme-linked immunosorbent assay (ELISA) kits for IL-17A, IL-6, IL-21 and TGF-β were purchased from BioLegend (San Diego, CA, USA). Fluorescence-activated cell sorting (FACS) human antibodies, including anti-CD3-phycoerythrin (PE)-Cy7 (Cat. no. 557851), anti-CD8-allophycocyanin (Cat. no. 561952), anti-CD4-fluorescein isothiocyanate (Cat. no. 561005), anti-Foxp3-PE (Cat. no. 560852), anti-IL-17A-PE (Cat. no. 560436), anti-IL-17A-PE-Cy7 (Cat. no. 560799) and their matched anti-mouse IgG1 K-PE (Cat. no. 551436)/PE-Cy7 (Cat. no. 552811) were all purchased from BD Biosciences (San Jose, CA, USA). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) reagents were obtained from Takara (Beijing, China). Rituximab was purchased from Novartis (Basel, Switzerland).
5
T-cell Cytokine Profiling in PBMC
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PBMC were incubated with medium in presence of CD28 (1 μg/ml) and CD49d (1 μg/ml) mAbs in a 200 μl final volume at 37 °C, 5% CO2 for one hour, followed by five-hour incubation in the presence of brefeldin A (GolgiPlug, BD). After a total of six hours incubation, cells were for surface and intracellular staining. In surface staining, cells were incubated with CD3-PBCD4-BV510, CD8-AF700, Vδ2-FITC solutions for 30 min at 4 °C without Ag stimulation in vitro. And then, cells were washed, fixed, permeabilized with FACS perm buffer (PBS supplemented with 0.5% Bovine Serum Albumin-BSA, 0.5% of saponin and 0.1% sodium azide) and incubated in solutions with anti-IFN-γ-BV711, anti-TNF-α-PE-CY7 and anti-IL-17A-PE (BD Biosciences - San Jose, CA, USA) for 30 min at 4 °C. The preparations, then maintained in 200 μL of FACS fix solution until acquisition in a Becton Dickinson FACS calibur instrument (Additional file 5 ). Isotype IgG was used as negative controls for staining cytokines or surface markers.
6
Multiparametric Flow Cytometry Analysis of Liver Immune Subsets
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For the analysis of Th1, Th2, Th17 subsets, approximately 106 leukocytes isolated from liver were stimulated with 20 ng/ml PMA, 1 μg/ml ionomycin and 2 mmol/ml monensin (Sigma-Aldrich, St. Louis, MO, USA) for 4 h at 37°C in 5% CO2. For surface staining, the cells were collected and then incubated with anti-CD4-FITC for 30min. For intracellular staining, the cells were washed and fixed, permeabilized with Perm/Fix solution (eBioscience, San Diego, CA, USA) for 30 min and stained with anti- IFN-γ- Alexa Flour 488, anti-IL-4- APC and anti- IL-17A-PE (BD Biosciences) for 30 min for detecting Th1, Th2, Th17 cells, respectively. All samples were evaluated on flow cytometer (FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA) using Diva software (BD Biosciences).
For detection of Treg cells, the liver leukocytes were first surface incubated with anti-CD4-PerCP-Cyanine5.5 and anti-CD25-FITC for 30 min. Subsequently, Foxp3 staining buffer set (eBioscience, San Diego, CA, USA) were used to treat the cells and anti-Foxp3-APC were added for 1 hour. All samples were evaluated on flow cytometer (FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA) using Diva software (BD Biosciences). Samples were acquired and analyzed on a FACSCanto II flow cytometer.
For detection of Treg cells, the liver leukocytes were first surface incubated with anti-CD4-PerCP-Cyanine5.5 and anti-CD25-FITC for 30 min. Subsequently, Foxp3 staining buffer set (eBioscience, San Diego, CA, USA) were used to treat the cells and anti-Foxp3-APC were added for 1 hour. All samples were evaluated on flow cytometer (FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA) using Diva software (BD Biosciences). Samples were acquired and analyzed on a FACSCanto II flow cytometer.
7
Quantification of Th1, Treg, and Th17 Cells by Flow Cytometry
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Mouse spleens were collected, ground and filtered through a sieve to obtain single cell suspensions. To detect Th1, Treg and Th17, cells were stimulated with phorbol myristate acetate (PMA)/ionomycin mixture and GolgiPlug (BD Biosciences) for 4h. Anti-CD4-FITC (BD Biosciences, 553046), Anti-CD3e APC-Cy7 (BD Biosciences, 557596) and Anti-CD25-BV421 (BD Biosciences, 607180) were used to stain the surface markers. After washing, cells were fixed and permeated using the Fixation/Permeabilization Kits (eBioscience). Anti-Foxp3-Alexa 647 (BD Biosciences, 560401), Anti-IFNG-PE-Cy7 (BD Biosciences, 557649), Anti-IL17A-PE (BD Biosciences, 559502) antibody was used for staining of intracellular markers. Finally, stained cells were detected by flow cytometry (FACSAriaIII, BD Biosciences) and data were analyzed with Flowjo software.
8
Profiling immune cell responses in atopic dermatitis
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PBMC and skin samples were obtained from patients with AD from the Department of Dermatology or from healthy donors from the Department of Plastic Surgery. This study was approved by the Ethical Committee from the University Hospital of Lille. All subjects provided written informed consent. Patients with AD were selected according to the Hanifin and Rajka criteria (Hanifin and Rajka, 1980 ). PBMCs were obtained after elimination of granulocytes on a Ficoll gradient. Punch skin biopsies (diameter 5 mm) were cultured in complete RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin (all Invitrogen), 5% human serum (Sigma-Aldrich), and 20 U IL-2/ml (Novartis). After 10–13 d, emigrating cells were collected and characterized by surface and intracellular staining by flow cytometry. Surface and intracellular cytokine staining was performed using the Cytofix/Cytoperm kit (BD) according to the manufacturer’s instructions. The following fluorochrome-conjugated antibodies were used: anti–IFN-γ–V450 (B27), anti-TNF–Alexa Fluor 700 (Mab11), anti–IL-4–PerCP-Cy5.5 (8D4-8), anti-IL17A-PE (N49-653), anti–IL-5–APC (TRFK5), anti–IL-9 PerCP-Cy5.5 (MH9A3), anti-CD4-APC-Cy7 (RPA-T4), anti–IL-13–Horizon-V450 (JES10-5A2; all BD), anti-IL4-PE (3010.211; BD), anti-IL22-APC (142928; R&D Systems), and CX3CR1-FITC (2A9-1; BioLegend).
9
Multicolor Flow Cytometry Antibody Panel
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The following fluorophore-conjugated antibodies were purchased from BD Biosciences: anti-CD4 Alx700 (RM4-5), anti-CD4 Alx647 (RM4-5), anti-CD8 PerCP (53–6.7), anti-CD11a (LFA-1) PE-Cy7 (2D7), anti-CD90.1 (Thy1.1) APC-Cy7 (OX-7), anti-CD127 Alx700 (A7R34), anti-CD62L PE (Mel-14), anti-CD44 APC (IM7), anti-IFN-γ APC, and anti-IL-17A PE. Anti-CD25 APC (PC61.5) and anti-Foxp3 PE (FJK-16s) were purchased from eBioscience, Inc. Goat pAb anti-GFP-FITC was purchased from Abcam. Anti-Fcγ-R (2.4G2) was produced from a hybridoma.
10
Phenotyping Cell Differentiation via Flow Cytometry
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In order to determine the different phenotypes of cell differentiation, flow cytometry was used. PBMCs and splenocytes from CIA mice, as well as cultured Th17 cells in vitro, were collected. Cells were then washed twice with PBS and activated with leukocyte activation cocktail (BD Pharmingen) for 4 h. After staining of Th17 cells with anti-CD4-FITC (BD Pharmingen) and anti-IL-17A-PE (BD Pharmingen) for 30 min, intracellular cytokines were analyzed by flow cytometry (Beckman FC-500, Beckman Coulter, Inc., Brea, CA, USA).
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