Journal: Immunology letters

Article Title: A lack of confirmation with alternative assays questions the validity of IL-17A expression in human neutrophils using immunohistochemistry.

doi: 10.1016/j.imlet.2014.10.025

Figure Lengend Snippet: Fig. 1. Detection of IL-17A in human neutrophils by IHC. (A) Cyanine3 labelled IL-17A+ cells infiltrate Wolbachia-containing nodules but not Wolbachia-depleted nodules. (B) Counterstaining

Article Snippet: Immunoprecipitation was carried out using 100 g mouse anti-human IL-17A antibody (41802, R&D Systems) and 60 g goat anti-human IL-17A antibody (AF-317-NA, R&D Systems) coupled to 1 mL HiTrap NHS-activated HP columns and AKTAprime Plus low-pressure affinity chromatography system (both from GE Healthcare) using the manufacturer’s protocol.

Techniques:

Journal: Immunology letters

Article Title: A lack of confirmation with alternative assays questions the validity of IL-17A expression in human neutrophils using immunohistochemistry.

doi: 10.1016/j.imlet.2014.10.025

Figure Lengend Snippet: Fig. 2. Determination of IL-17A expression by human neutrophils. (A) Western blotting of neutrophil (PMN) cell lysate using goat anti-IL-17A antibody and mouse anti- IL-17A antibody.

Article Snippet: Immunoprecipitation was carried out using 100 g mouse anti-human IL-17A antibody (41802, R&D Systems) and 60 g goat anti-human IL-17A antibody (AF-317-NA, R&D Systems) coupled to 1 mL HiTrap NHS-activated HP columns and AKTAprime Plus low-pressure affinity chromatography system (both from GE Healthcare) using the manufacturer’s protocol.

Techniques: Expressing, Western Blot

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Interleukin-17A expression in human synovial mast cells in rheumatoid arthritis and osteoarthritis.

doi: 10.1016/j.alit.2016.04.007

Figure Lengend Snippet: Fig. 1. Expression of IL-17A in synovial MCs from OA patients and from RA patients. Representative images of IL-17Aþ MCs (tryptaseþ cells) in synovia from an OA patient (A) and an RA patient (B). Immunochemical staining of synovia with anti-tryptase mAb (green, a) and anti-IL-17A (red, b) Ab, and a merged image (yellow, c). The cells were stained with DAPI (blue). The white arrow heads indicate tryptase-IL-17A double-positive cells. Bar ¼ 10 mm. (C) As negative controls, a synovium sample from an OA patient was stained with mouse IgG1 and goat IgG. The results are representative of separate analyses using 6 patients with OA and 6 patients with RA.

Article Snippet: The following antibodies (Abs) were purchased from the indicated sources: human IgE (Calbiochem, San Diego, CA, USA); goat anti-human IL-17A polyclonal Ab (R&D Systems, Minneapolis, MN, USA); anti-human tryptase monoclonal (m) Ab (clone AA1; DakoCytomation Inc., Carpinteria, CA, USA); anti-Fc 3RIa mAb (clone CRA1; eBioscience, San Diego, CA, USA); anti-human IgE Ab (DakoCytomation Inc.); and F(ab0)2 fragments of anti-human FcgRI (clone 10.1; ID Labs Inc., London, ON, Canada).

Techniques: Expressing, Staining

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Interleukin-17A expression in human synovial mast cells in rheumatoid arthritis and osteoarthritis.

doi: 10.1016/j.alit.2016.04.007

Figure Lengend Snippet: Fig. 2. A comparison of the numbers of MCs (A), the numbers of IL-17Aþ MCs (B), the numbers of IL-17Aþ cells (C), the percentage of IL-17Aþ MCs among all the MCs (D), and the percentage of IL-17Aþ MCs among all the IL-17Aþ cells (E) in synovia from OA patients (open circles, n ¼ 6) and from RA patients (closed circles, n ¼ 6). Data are expressed as the median and inter-quartile range. N.S., not significant.

Article Snippet: The following antibodies (Abs) were purchased from the indicated sources: human IgE (Calbiochem, San Diego, CA, USA); goat anti-human IL-17A polyclonal Ab (R&D Systems, Minneapolis, MN, USA); anti-human tryptase monoclonal (m) Ab (clone AA1; DakoCytomation Inc., Carpinteria, CA, USA); anti-Fc 3RIa mAb (clone CRA1; eBioscience, San Diego, CA, USA); anti-human IgE Ab (DakoCytomation Inc.); and F(ab0)2 fragments of anti-human FcgRI (clone 10.1; ID Labs Inc., London, ON, Canada).

Techniques: Comparison

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Interleukin-17A expression in human synovial mast cells in rheumatoid arthritis and osteoarthritis.

doi: 10.1016/j.alit.2016.04.007

Figure Lengend Snippet: Fig. 3. IL-17A production from cultured synovium-derived MC following IgE- and IgG-dependent stimulation. (A) IgE-sensitized MCs from patients with OA (white bars, n ¼ 4 donors) or from patients with RA (black bars, n ¼ 4 donors) were incubated with anti-IgE for 6 h. (B) MCs from patients with OA (n ¼ 4 donors) were incubated with 1 mg/ml of F(ab0)2aFcgRI (black bars) or F(ab0)2mIgG1 (white bars) for 30 min and were then stimulated with gF(ab0)2amF(ab0)2 for 6 h. (C) MCs from patients with OA (n ¼ 6 donors) were stimulated with monomeric IgG or aggregated IgG for 6 h. (D) MCs from patients with OA (white bars, n ¼ 7 donors) and from patients with RA (black bars, n ¼ 4 donors) were stimulated with IL-33 for 6 h. (E, F) MCs from patients with OA (n ¼ 5e6 donors) were stimulated with anti-Fc

Article Snippet: The following antibodies (Abs) were purchased from the indicated sources: human IgE (Calbiochem, San Diego, CA, USA); goat anti-human IL-17A polyclonal Ab (R&D Systems, Minneapolis, MN, USA); anti-human tryptase monoclonal (m) Ab (clone AA1; DakoCytomation Inc., Carpinteria, CA, USA); anti-Fc 3RIa mAb (clone CRA1; eBioscience, San Diego, CA, USA); anti-human IgE Ab (DakoCytomation Inc.); and F(ab0)2 fragments of anti-human FcgRI (clone 10.1; ID Labs Inc., London, ON, Canada).

Techniques: Cell Culture, Derivative Assay, Incubation

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Interleukin-17A expression in human synovial mast cells in rheumatoid arthritis and osteoarthritis.

doi: 10.1016/j.alit.2016.04.007

Figure Lengend Snippet: Fig. 4. IL-17A (A) and IL-8 production (B) from cultured synovium-derived MC following various stimulations. MCs from patients with OA (n ¼ 3 donors) were stimulated with TNF- a, C5a, LPS, or IL-23 plus IL-1b for 24 h. IL-17 levels were under the detection limit in 2 donors out of 3 donors. The data of IL-17A production from one donor are shown. The data of IL-8 production are shown as the mean ± SEM. *P < 0.05.

Article Snippet: The following antibodies (Abs) were purchased from the indicated sources: human IgE (Calbiochem, San Diego, CA, USA); goat anti-human IL-17A polyclonal Ab (R&D Systems, Minneapolis, MN, USA); anti-human tryptase monoclonal (m) Ab (clone AA1; DakoCytomation Inc., Carpinteria, CA, USA); anti-Fc 3RIa mAb (clone CRA1; eBioscience, San Diego, CA, USA); anti-human IgE Ab (DakoCytomation Inc.); and F(ab0)2 fragments of anti-human FcgRI (clone 10.1; ID Labs Inc., London, ON, Canada).

Techniques: Cell Culture, Derivative Assay

Journal: Immune Network

Article Title: Expanded IL-22 + Group 3 Innate Lymphoid Cells and Role of Oxidized LDL-C in the Pathogenesis of Axial Spondyloarthritis with Dyslipidaemia

doi: 10.4110/in.2021.21.e43

Figure Lengend Snippet: Correlation between circulating immune cells and ASDAS-CRP. ILC3 cells in patients with axSpA (n=34) were analysed by flow cytometry. Spearman correlation coefficients were calculated between circulating IL-22 + ILC3, IL-17A + NKp44 - ILC3, IFN-γ + CD4 + cells, IL-17A + CD4 + cells, IL-22 + CD4 + cells, and ASDAS-CRP.

Article Snippet: Briefly, a 96-well plate (Eppendorf, Hamburg, Germany) was coated with monoclonal Abs against TNF-α (DY210; R&D systems, Minneapolis, MN, USA), IL-17A (DY317; R&D systems), and IL-22 (MAB7822, 782-IL, BAM7821; R&D systems) at 4°C overnight.

Techniques: Flow Cytometry

Journal: STAR Protocols

Article Title: Simplified mass cytometry protocol for in-plate staining, barcoding, and cryopreservation of human PBMC samples in clinical trials

doi: 10.1016/j.xpro.2022.101362

Figure Lengend Snippet: Intracellular antibody master mix

Article Snippet: Anti-Human IL-17A (N49-653)-164Dy—100 Tests (1:100) , Fluidigm , Cat#3164002B.

Techniques:

Journal: STAR Protocols

Article Title: Simplified mass cytometry protocol for in-plate staining, barcoding, and cryopreservation of human PBMC samples in clinical trials

doi: 10.1016/j.xpro.2022.101362

Figure Lengend Snippet:

Article Snippet: Anti-Human IL-17A (N49-653)-164Dy—100 Tests (1:100) , Fluidigm , Cat#3164002B.

Techniques: Recombinant, Saline, Staining, Blocking Assay, Isolation, Software, Cell Counting, Cytometry

Journal: Frontiers in Immunology

Article Title: Age-related increase of mitochondrial content in human memory CD4+ T cells contributes to ROS-mediated increased expression of proinflammatory cytokines

doi: 10.3389/fimmu.2022.911050

Figure Lengend Snippet: Capacity of cytokine production in memory CD4 + T cell. Cells were activated with PMA/ionomycin for 5 h, adding BFA for the last 3 hours for intracellular cytokine expression. (A) Representative example of cytokine expression in memory CD4 + T cells from young and aged donors after stimulation with PMA/ionomycin. (B) Frequencies in % and geometric mean fluorescence intensity (gMFI) of IL-2, IL-4, IL-10, IL-17, IFN-γ, and TNF-α producing memory CD4 + T cells (mean ± SEM; n = 7).

Article Snippet: Incubation was conducted by dissolving 1x 10 6 cells/ml in RPMI 1640 medium (Thermo Fisher Scientific) with 5% human AB serum (Sigma Aldrich) and 1% penicillin/streptomycin (Thermo Fisher Scientific) at 37˚C in a 5% CO 2 atmosphere Cells were collected, washed, fixed, permeabilized (Inside staining kit, Miltenyi Biotec) and stained with a combination of monoclonal antibodies including anti-human-IFNγ (PERCP-Cy5.5, Biolegend, clone 4SB3, Cat.# 502526), anti-human-IL-2 (APC-vio770, Miltenyi Biotec, clone N7.48 A, Cat.# 130-097-011), anti-human-IL-4 (PE, Miltenyi Biotec, clone 7A3-3, Cat.# 130-091-647), anti-human-IL-17A (FITC, Miltenyi Biotec, clone CZ8-23G1, Cat.# 130-094-520), and anti-human-TNF-α (PE-Vio770 (Miltenyi Biotec, clone cA2, Cat.# 130-096-755).

Techniques: Expressing, Fluorescence

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Alteration of Th17 and Treg cells in patients with unexplained recurrent spontaneous abortion before and after lymphocyte immunization therapy

doi: 10.1186/1477-7827-12-74

Figure Lengend Snippet: The percentages of Th17 and Treg cells in peripheral blood mononuclear cells and the Th17/Treg ratio varied with immunotherapy. The percentage of Th17 and Treg cells in peripheral blood mononuclear cells were detected by flow cytometry in the total patient population before and after immunotherapy, respectively. (a) The percentage of Th17 cells in peripheral blood mononuclear cells significantly decreased after immunotherapy (P < 0.01, paired t -test). Bar represents the CD4 + /IL-17A + cell frequency and values of the means ± S.E. The percentage of Treg cells in peripheral blood mononuclear cells significantly increased after immunotherapy (P < 0.01, paired t -test). Bar represents the frequency of CD4 + /CD25bright/Foxp3 + cells and the means ± S.E. are shown. (b) The Th17/Treg ratio in peripheral blood mononuclear cells significantly decreased after immunotherapy (P < 0.01, paired t -test). Bar represent Th17/Treg ratio in total patients before and after immunotherapy and values of means ± S.E. Representative CD4+/IL-17A + flow cytometry plots in patients are shown before (c) and after (d) immunology. Representative CD4+/CD25bright/Foxp3+ flow cytometry plots in patients are shown before (e) and after (f) immunology.

Article Snippet: For Th17 analysis, the cells were incubated with fluorescein isothiocyanate (FITC) anti-human CD4 (eBioscience, San Diego, CA, USA) at 4°C for 20 min. For Treg analysis, the cells were incubated with PerCP anti-human CD4 (eBioscience, USA) and FITC anti-human CD25 (eBioscience, San Diego, CA, USA) at 4°C for 20 min. After the surface staining, the cells were then fixed and permeabilized with Perm/Fix solution (Beckman Coulter) and were stained with PE anti-human IL-17A (eBioscience) for Th17 detection or PE anti-human Foxp3 (eBioscience, CA, USA) for Treg detection.

Techniques: Flow Cytometry

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Alteration of Th17 and Treg cells in patients with unexplained recurrent spontaneous abortion before and after lymphocyte immunization therapy

doi: 10.1186/1477-7827-12-74

Figure Lengend Snippet: The change of Th17 and Treg cells in peripheral blood mononuclear cells and the Th17/Treg ratio varies in total patients and patients with a successful pregnancy after immunotherapy. The detailed variation of CD4+/IL-17A + (a) , CD4+/CD25bright/Foxp3+ (b) and Th17/Treg ratio (c) in each patient before and after immunotherapy is shown in line chart. The detailed variation of CD4+/IL-17A + (d) , CD4+/CD25bright/Foxp3 + (e) and Th17/Treg ratio (f) in each patient with a successful pregnancy after immunotherapy is shown in the line chart.

Article Snippet: For Th17 analysis, the cells were incubated with fluorescein isothiocyanate (FITC) anti-human CD4 (eBioscience, San Diego, CA, USA) at 4°C for 20 min. For Treg analysis, the cells were incubated with PerCP anti-human CD4 (eBioscience, USA) and FITC anti-human CD25 (eBioscience, San Diego, CA, USA) at 4°C for 20 min. After the surface staining, the cells were then fixed and permeabilized with Perm/Fix solution (Beckman Coulter) and were stained with PE anti-human IL-17A (eBioscience) for Th17 detection or PE anti-human Foxp3 (eBioscience, CA, USA) for Treg detection.

Techniques:

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Alteration of Th17 and Treg cells in patients with unexplained recurrent spontaneous abortion before and after lymphocyte immunization therapy

doi: 10.1186/1477-7827-12-74

Figure Lengend Snippet: The percentage of Th17 and Treg cells in peripheral blood mononuclear cells and the Th17/Treg ratio varies in patients with a successful pregnancy after immunotherapy. The percentage of Th17 and Treg cells in peripheral blood mononuclear cells were detected by flow cytometry in patients with successful pregnancy after immunotherapy. To date, 13 of the 20 patients have become newly pregnant. Of the 13, the 11 patients did not miscarry (successful group). (a) The percentage of Th17 cells in peripheral blood mononuclear cells significantly decreased after immunotherapy. The percentage of Treg cells in peripheral blood mononuclear cells significantly increased after immunotherapy (P < 0.01, paired t -test). Bar represents the CD4+/IL-17A + cell frequency and the means ± S.E. are indicated (b) The Th17/Treg ratio in peripheral blood mononuclear cells significantly decreased after immunotherapy (P < 0.01, paired t -test). Bar represent the frequency of CD4+/CD25bright/Foxp3+ cells and the means ± S.E. are indicated. Representative CD4+/IL-17A + flow cytometry plots from patients with successful pregnancies were shown before (c) and after (d) immunotherapy. Representative CD4+/CD25bright Foxp3+ flow cytometry plots from patients with successful pregnancies were shown before (e) and after (f) immunotherapy.

Article Snippet: For Th17 analysis, the cells were incubated with fluorescein isothiocyanate (FITC) anti-human CD4 (eBioscience, San Diego, CA, USA) at 4°C for 20 min. For Treg analysis, the cells were incubated with PerCP anti-human CD4 (eBioscience, USA) and FITC anti-human CD25 (eBioscience, San Diego, CA, USA) at 4°C for 20 min. After the surface staining, the cells were then fixed and permeabilized with Perm/Fix solution (Beckman Coulter) and were stained with PE anti-human IL-17A (eBioscience) for Th17 detection or PE anti-human Foxp3 (eBioscience, CA, USA) for Treg detection.

Techniques: Flow Cytometry

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Alteration of Th17 and Treg cells in patients with unexplained recurrent spontaneous abortion before and after lymphocyte immunization therapy

doi: 10.1186/1477-7827-12-74

Figure Lengend Snippet: The interleukins/cytokines were detected by multiplex fluorochrome assay in serum samples from the total patients before and after immunotherapy. (a) The content of TGF-β, IL-10, IFN-γ, TNF-α, IL-17A, IL-23, and IL-6 in serum varied before and after immunotherapy. The detailed variation of TGF-β (b) , IL-10 (c) , IFN-γ (d) , TFN-a (e) , IL-17A (f) , IL-23 (g) , and IL-6 (h) in each patient before and after immunotherapy is shown in the line chart.

Article Snippet: For Th17 analysis, the cells were incubated with fluorescein isothiocyanate (FITC) anti-human CD4 (eBioscience, San Diego, CA, USA) at 4°C for 20 min. For Treg analysis, the cells were incubated with PerCP anti-human CD4 (eBioscience, USA) and FITC anti-human CD25 (eBioscience, San Diego, CA, USA) at 4°C for 20 min. After the surface staining, the cells were then fixed and permeabilized with Perm/Fix solution (Beckman Coulter) and were stained with PE anti-human IL-17A (eBioscience) for Th17 detection or PE anti-human Foxp3 (eBioscience, CA, USA) for Treg detection.

Techniques: Multiplex Assay

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Alteration of Th17 and Treg cells in patients with unexplained recurrent spontaneous abortion before and after lymphocyte immunization therapy

doi: 10.1186/1477-7827-12-74

Figure Lengend Snippet: The ratio of IFN-γ/IL-10, and IL-17A/TGF-βvaried in total patients before and after immunotherapy. The detailed variation of the ratio of IFN-γ/IL-10 (a) , and IL-17A/TGF-β (b) in each patient is shown in the line chart.

Article Snippet: For Th17 analysis, the cells were incubated with fluorescein isothiocyanate (FITC) anti-human CD4 (eBioscience, San Diego, CA, USA) at 4°C for 20 min. For Treg analysis, the cells were incubated with PerCP anti-human CD4 (eBioscience, USA) and FITC anti-human CD25 (eBioscience, San Diego, CA, USA) at 4°C for 20 min. After the surface staining, the cells were then fixed and permeabilized with Perm/Fix solution (Beckman Coulter) and were stained with PE anti-human IL-17A (eBioscience) for Th17 detection or PE anti-human Foxp3 (eBioscience, CA, USA) for Treg detection.

Techniques:

Journal: Mediators of Inflammation

Article Title: Analysis of Th17 and Tc17 Frequencies and Antiviral Defenses in Gut-Associated Lymphoid Tissue of Chronic HIV-1 Positive Patients

doi: 10.1155/2015/395484

Figure Lengend Snippet: Activation markers, IFN- γ and IL-17A expression in CD4 + and CD8 + T cells subsets in blood and in gut. Panel (a): representative flow cytometry plots outlining CD38 and HLA-DR expression on naïve, effector, and central memory CD4 + and CD8 + lymphocytes derived from peripheral blood mononuclear cells (PBMCs) and gut-associated lymphoid tissue (GALT). Panel (b): representative flow cytometry plots outlining IFN- γ and IL-17A intracitoplasmatic expression on naïve, effector, and central memory CD4 + (Th1 and Th17) and CD8 + (Tc1 and Tc17) lymphocytes derived from PBMCs and GALT.

Article Snippet: Cultured cells were fixed, permeabilized (BD Cytofix/Cytoperm, Becton Dickinson, San Jose, CA, USA), and stained with combinations of fluorochrome-labeled monoclonal antibodies: CD3-PerCP, CD4-APC-Vio770, CD8-FITC, CD45RO-PE-Vio770, CD27-VioBlue, IFN- γ -APC, and IL-17A-PE (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activation Assay, Expressing, Flow Cytometry, Derivative Assay