flow cytometry based il 22 secretion assay detection kit pe  (Miltenyi Biotec)

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: The herbal extract EPs® 7630 increases the antimicrobial airway defense through monocyte-dependent induction of IL-22 in T cells

doi: 10.1007/s00109-020-01970-3

Figure Lengend Snippet: EPs® 7630 stimulates IL-22 and IL-17 secretion of human PBMCs. a Human PBMCs were stimulated in a kinetic approach with 3 μg/ml EPs® 7630, 100 ng/ml LPS, or were left untreated as indicated. b Dose-response analysis was performed by treatment of human PBMCs with increasing concentrations (0–3 μg/ml, as indicated) of EPs® 7630 for 48 h. Quantification of IL-22, IL-17, and IFN-γ in culture supernatants was performed by ELISA. Data from 3 ( a ) or 12 ( b ) independent experiments are given as mean ± SEM. Significant differences among treatment groups are indicated (* p < 0.05; ** p < 0.01, Wilcoxon matched-pairs signed-rank test)

Article Snippet: To characterize EPs® 7630–dependent IL-22 producers, an IL-22-specific secretion assay (Miltenyi Biotec) was performed using PBMCs according to the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: The herbal extract EPs® 7630 increases the antimicrobial airway defense through monocyte-dependent induction of IL-22 in T cells

doi: 10.1007/s00109-020-01970-3

Figure Lengend Snippet: EPs® 7630 pretreatment modulates the cytokine production of activated lymphocytes. a , b Human PBMCs were pretreated with different concentrations of EPs® 7630 as indicated for 24 h. Afterwards, activators of T cells (anti-CD3/anti-CD28 Abs) ( a ) and innate lymphoid cells (cytokine mixture composed of IL-1β, IL-2, and IL-12) ( b ) were added for further 24 h. Quantification of IL-17, IL-22 and IFN-γ in culture supernatants was performed by ELISA. Data from 12 donors per group are given as mean ± SEM. Significant differences are indicated (* p < 0.05; ** p < 0.01, Wilcoxon matched-pairs signed-rank test)

Article Snippet: To characterize EPs® 7630–dependent IL-22 producers, an IL-22-specific secretion assay (Miltenyi Biotec) was performed using PBMCs according to the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: The herbal extract EPs® 7630 increases the antimicrobial airway defense through monocyte-dependent induction of IL-22 in T cells

doi: 10.1007/s00109-020-01970-3

Figure Lengend Snippet: IL-22 strengthens the antibacterial defense of airway epithelial cells. a A549 human lung epithelial cells were cultured in the presence or absence (control) of IL-22, its inhibitor IL-22BP, or a combination of IL-22 and IL-22BP for 48 h. b BALB/c mice were i.p. injected with PBS (control) or IL-22. At the indicated time points after injection, mice were sacrificed and lung tissue was taken for analysis of S100A9 expression by RT-qPCR. Data of 4 (0 h control and PBS) or 3 (IL-22) mice per group are given as mean ± SEM. c A549 human lung epithelial cells were pretreated or not with 3 μg/ml EPs® 7630 for 24 h followed by stimulation with IL-22, IL-17A, IFN-γ, or the combination of IL-17A and IL-22 for 48 h or were left unstimulated (control). a , c Expression of S100A9, LCN2, and MX1 was analyzed by RT-qPCR. Data of 7 ( a ) or 3–4 ( c ) independent experiments are given as mean ± SEM. Significant differences among treatment groups are indicated (* p < 0.05, Wilcoxon matched-pairs signed-rank test)

Article Snippet: To characterize EPs® 7630–dependent IL-22 producers, an IL-22-specific secretion assay (Miltenyi Biotec) was performed using PBMCs according to the manufacturer’s protocol.

Techniques: Cell Culture, Injection, Expressing, Quantitative RT-PCR

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: The herbal extract EPs® 7630 increases the antimicrobial airway defense through monocyte-dependent induction of IL-22 in T cells

doi: 10.1007/s00109-020-01970-3

Figure Lengend Snippet: CD4 + memory T cells are no direct targets of EPs® 7630. a CD4 + memory T cells were isolated from human PBMCs by magnetic labeling–based cell sorting and cultured for 48 h in the absence (control) or presence of EPs® 7630 (0–10 μg/ml) as indicated. b CD4 + memory T cells were stimulated as described in a and were further activated after 24 h of culture by anti-CD3/CD28 antibodies (Dynabeads) for another 24 h. a , b Culture supernatants were analyzed for IL-22, IL-17, and IFN-γ level by ELISA. Data from 5 independent experiments are given as mean ± SEM. Significant differences among treatment groups are indicated (* p < 0.05, Wilcoxon matched-pairs signed-rank test)

Article Snippet: To characterize EPs® 7630–dependent IL-22 producers, an IL-22-specific secretion assay (Miltenyi Biotec) was performed using PBMCs according to the manufacturer’s protocol.

Techniques: Isolation, Labeling, FACS, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: The herbal extract EPs® 7630 increases the antimicrobial airway defense through monocyte-dependent induction of IL-22 in T cells

doi: 10.1007/s00109-020-01970-3

Figure Lengend Snippet: Monocytes play a key role in EPs® 7630–induced IL-22 production by T cells. a CD4 + memory T cells and autologous monocytes were co-cultured or cultured alone for 72 h in the absence (control) or presence of 10 μg/ml EPs® 7630 (EPs® 7630). b CD4 + memory T cells and autologous monocytes were pretreated in separate cultures with EPs® 7630 (10 μg/ml) or medium with solvent for 24 h. Afterwards, CD4 + memory T cells and monocytes were washed and co-cultured as indicated for 72 h without further EPs® 7630 stimulation. c CD4 + memory T cells were cultured for 72 h in the presence of supernatant (SN) obtained from cultures of EPs® 7630–stimulated (10 μg/ml) monocytes. d CD4 + memory T cells and autologous monocytes were co-cultured with (no transwell) or without enabled cell-cell contact (transwell) or were cultured separately for 72 h in the presence of 10 μg/ml EPs® 7630. a – d Human CD4 + memory T cells and autologous monocytes were each isolated by magnetic labeling–based cell sorting. Quantification of IL-22 in culture supernatants was carried out by ELISA. e Human PBMCs were stimulated or not (solvent control) in a kinetic approach with 10 μg/ml EPs® 7630 or 100 ng/ml LPS or were left without stimulation up to 72 h. Quantification of IL-1β, IL-23, and IL-2 levels in culture supernatant was performed by ELISA. f Human PBMCs were stimulated or not (solvent control) with 3 μg/ml EPs® 7630, in the presence of 1.5 μg/ml IL-1RA, 3 μg/ml anti-IL-23p19 antibody, or a combination thereof for 72 h. Quantification of IL-22 in culture supernatants was carried out by ELISA. Data from 6 ( a ), 2 ( b ), 4 ( c ), 5 ( d ) 3–4 ( e ), and 5 ( f ) independent experiments are given as mean ± SEM. Significant differences between treatment groups are indicated (* p < 0.05, Wilcoxon matched-pairs signed-rank test). Tmem: CD4 + memory T cells; mo: monocytes

Article Snippet: To characterize EPs® 7630–dependent IL-22 producers, an IL-22-specific secretion assay (Miltenyi Biotec) was performed using PBMCs according to the manufacturer’s protocol.

Techniques: Cell Culture, Solvent, Isolation, Labeling, FACS, Enzyme-linked Immunosorbent Assay

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: The herbal extract EPs® 7630 increases the antimicrobial airway defense through monocyte-dependent induction of IL-22 in T cells

doi: 10.1007/s00109-020-01970-3

Figure Lengend Snippet: EPs® 7630 induces IL-22 secretion by different CD4 + memory T cell subsets. a – c Human PBMCs were stimulated or not (solvent control) with 10 μg/ml EPs® 7630 for 72 h as indicated. Afterwards, an IL-22-specific secretion assay and surface marker staining was performed followed by flow cytometry–based analysis. a Schematic overview of the experimental setting of IL-22-specific secretion assay. b Data from one representative out of three independent experiments are given. c Data from 3 independent experiments are given as mean ± SEM

Article Snippet: To characterize EPs® 7630–dependent IL-22 producers, an IL-22-specific secretion assay (Miltenyi Biotec) was performed using PBMCs according to the manufacturer’s protocol.

Techniques: Solvent, Marker, Staining, Flow Cytometry