goat anti gal 9  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Galectin-9 Regulates Monosodium Urate Crystal-Induced Gouty Inflammation Through the Modulation of Treg/Th17 Ratio

doi: 10.3389/fimmu.2021.762016

Figure Lengend Snippet: Gal-9 attenuates MSU crystals-induced gouty inflammation in mouse knee joints. Mice were treated with intra-articular (i.a.) dose of Gal-9 (9μg/20μl) or vehicle (PBS, 20μl) 30 mins after i.a. stimulation with MSU crystals (200μg/20μl) in the right knee joints. (A) Joint inflammation score (0-3 in increments of 0.25) and (B) joint inflammation oedema was evaluated at 4, 18, 24 and 48h after the stimulus with MSU. Data (expressed as joint inflammation score and Δ increase of knee joints mm respectively) are presented as means ± SEM of n = 6 mice per group. Statistical analysis was conducted by one- or two-way ANOVA followed by Bonferroni’s for multiple comparisons. ## P ≤ 0.01, #### P ≤ 0.0001 vs Ctrl group; **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 vs MSU group.

Article Snippet: Cells were washed twice and incubated with anti-human Gal-9 antibody goat IgG (1:200, R&D Systems) and stained with the following conjugated antibodies (BioLegend, London, UK): CD4 (1:50, clone RPAT4), donkey anti-goat IgG (1:200, polyclonal) for 20 mins at 4°C.

Techniques:

Journal: Frontiers in Immunology

Article Title: Galectin-9 Regulates Monosodium Urate Crystal-Induced Gouty Inflammation Through the Modulation of Treg/Th17 Ratio

doi: 10.3389/fimmu.2021.762016

Figure Lengend Snippet: Gal-9 modulates the recruitment of innate immune cells. Flow cytometry analysis was employed to determine in situ neutrophil and monocyte subsets. At the peak of inflammatory reaction (18h), ankle joints were digested, and single cell suspensions were obtained. Flow cytometry strategy applied to identify neutrophils (A) , and monocytes (B) , after Gal-9 treatment are shown. Cells were washed and stained with: CD45, LY6-C, LY6-G, CD11b, and CD115/CD45 + cells were plotted for LY6-C and LY6-G expression to identify CD45 + /LY6-C hi /LY6-G hi as neutrophils (A) . CD11b + cells were plotted for LY6-C and CD115 expression to distinguish CD11b + /CD115 + /LY6-C lo patrolling monocytes from CD11b + /CD115 + /LY6-C hi inflammatory monocytes (B) . (C) Total infiltrated leukocytes, (D) neutrophil and (E) inflammatory monocytes were quantified in the different experimental conditions. Representative FACS plots of three independent experiments with similar results are shown. Values are presented as means ± SEM of n = 6 mice per group. Statistical analysis was conducted by one-way ANOVA followed by Bonferroni’s for multiple comparisons. ## P ≤ 0.01 vs Ctrl group; *P ≤ 0.05, **P ≤ 0.01 vs MSU group.

Article Snippet: Cells were washed twice and incubated with anti-human Gal-9 antibody goat IgG (1:200, R&D Systems) and stained with the following conjugated antibodies (BioLegend, London, UK): CD4 (1:50, clone RPAT4), donkey anti-goat IgG (1:200, polyclonal) for 20 mins at 4°C.

Techniques: Flow Cytometry, In Situ, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: Galectin-9 Regulates Monosodium Urate Crystal-Induced Gouty Inflammation Through the Modulation of Treg/Th17 Ratio

doi: 10.3389/fimmu.2021.762016

Figure Lengend Snippet: Gal-9 sustains local Treg levels. Flow cytometry analysis was employed to determine in situ levels of CD4 + T cells, Th17 and Tregs subsets. At the peak of the inflammatory reaction (18h), ankle joints were digested, and single cell suspensions were obtained. Flow cytometry strategy applied to identify the modulation of CD3/CD4 (A, B) , CD4 + T cells (A, C) , Th17 (A, C, D) and Tregs (A, C, E) after Gal-9 treatment are shown. Cells were washed and stained with: CD3, CD4, CD25, and intracellular antibodies IL-17A and FOXP-3. Th17 and Treg populations were defined as CD4 + /IL-17 + (A, C, D) and CD4 + /CD25 + /FOXP-3 + (A, C, E) respectively. (F) Total CD4 + T cells, (G) Th17 and (H) Tregs were quantified in the different experimental conditions. Representative FACS plots of three independent experiments with similar results are shown. Values are presented as means ± SEM of n = 6 mice per group. Statistical analysis was conducted by one-way ANOVA followed by Bonferroni’s test for multiple comparisons. # P ≤ 0.05, ## P ≤ 0.01 vs Ctrl group; *P ≤ 0.05 vs MSU group.

Article Snippet: Cells were washed twice and incubated with anti-human Gal-9 antibody goat IgG (1:200, R&D Systems) and stained with the following conjugated antibodies (BioLegend, London, UK): CD4 (1:50, clone RPAT4), donkey anti-goat IgG (1:200, polyclonal) for 20 mins at 4°C.

Techniques: Flow Cytometry, In Situ, Staining

Journal: Frontiers in Immunology

Article Title: Galectin-9 Regulates Monosodium Urate Crystal-Induced Gouty Inflammation Through the Modulation of Treg/Th17 Ratio

doi: 10.3389/fimmu.2021.762016

Figure Lengend Snippet: Gal-9 decreases the release of cyto-chemokines in knee joints. Inflammatory fluids obtained from knee joint homogenates at 18h time-point, were assayed using a proteome profiler cytokine array for (A) CTRL, (B) MSU and (C) MSU + Gal-9 group. Densitometric analysis is presented as heatmap (D) . Thereafter, IL-10, IL-17, KC, JE, MIP-1α and TNF-α were extrapolated from heatmap and represented graphically (E) . Data (expressed as INT/mm 2 ) are presented as means ± SEM. of positive spots of three separate independent experiments run each with n = 6 mice per group pooled. Statistical analysis was performed by using two-way ANOVA followed by Bonferroni’s test for multiple comparisons. ### P ≤ 0.001, #### P ≤ 0.0001 vs Ctrl group; *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 vs MSU group.

Article Snippet: Cells were washed twice and incubated with anti-human Gal-9 antibody goat IgG (1:200, R&D Systems) and stained with the following conjugated antibodies (BioLegend, London, UK): CD4 (1:50, clone RPAT4), donkey anti-goat IgG (1:200, polyclonal) for 20 mins at 4°C.

Techniques:

Journal: Frontiers in Immunology

Article Title: Galectin-9 Regulates Monosodium Urate Crystal-Induced Gouty Inflammation Through the Modulation of Treg/Th17 Ratio

doi: 10.3389/fimmu.2021.762016

Figure Lengend Snippet: Gal-9 induces Treg differentiation of naïve human CD4 + T cells. CD4 + T cells were isolated from PBMCs and incubated with 10nM Gal-9 before staining with anti-Gal-9. (A) Representative histogram showing exogenous Gal-9 binding in CD4 + cells compared to isotype control levels. (B) Quantification of Gal-9 protein levels using median fluorescence intensity (MFI) of Gal-9. Data are expressed as mean ± SEM (n=4). Statistical analysis was performed with Students T-test; *P < 0.05 vs BSA. Naïve CD4 + T cells were differentiated with activation cocktail (CD3/CD28, IL2, TGFβ) or Gal-9 alone for 5 days. (C, D) Flow cytometry was used to measure % positive expression of CD4 + ,CD25 + and Foxp3 + . (E) Cell culture supernatants were collected, and IL-10 levels were measured by ELISA. Data are expressed as mean ± SEM (n=3). Statistical analysis was performed with one-way ANOVA with Tukey multiple comparisons; *P < 0.05.

Article Snippet: Cells were washed twice and incubated with anti-human Gal-9 antibody goat IgG (1:200, R&D Systems) and stained with the following conjugated antibodies (BioLegend, London, UK): CD4 (1:50, clone RPAT4), donkey anti-goat IgG (1:200, polyclonal) for 20 mins at 4°C.

Techniques: Isolation, Incubation, Staining, Binding Assay, Fluorescence, Activation Assay, Flow Cytometry, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Tissue and plasma levels of galectins in patients with high grade serous ovarian carcinoma as new predictive biomarkers

doi: 10.1038/s41598-017-13802-5

Figure Lengend Snippet: Predictive value of circulating galectins on the OS and DFS. ( A) Gal-8 and ( B ) gal-9 concentration in the plasma of healthy donors and ovarian cancer patients. ( C ) Kaplan-Meier curves of the 5-years OS and 5-years DFS according to the presence of gal-8 and gal-9 in the plasma of HGSC patients. Blue bar: galectin negative samples, red bar: galectin positive samples.

Article Snippet: Rabbit anti-human gal-9 (1:100, Abcam), goat anti-human gal-9 (1:50, R&D Systems), rabbit anti-human LC3B (1:200, Sigma) and rabbit anti-human Cox IV (1:500, New England Biolabs, Ipswich, MA) primary antibodies were used.

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Tissue and plasma levels of galectins in patients with high grade serous ovarian carcinoma as new predictive biomarkers

doi: 10.1038/s41598-017-13802-5

Figure Lengend Snippet: Predictive value of circulating galectin-8 and -9 according to CA-125 levels. Kaplan-Meier curves of 5-years OS and 5-years DFS according to the presence of circulating gal-8 and gal-9 in the plasma of ( A ) CA125 LOW or ( B ) CA125 HIGH patients. Blue bar: galectin negative samples, red bar: galectin positive samples.

Article Snippet: Rabbit anti-human gal-9 (1:100, Abcam), goat anti-human gal-9 (1:50, R&D Systems), rabbit anti-human LC3B (1:200, Sigma) and rabbit anti-human Cox IV (1:500, New England Biolabs, Ipswich, MA) primary antibodies were used.

Techniques:

Journal: Scientific Reports

Article Title: Tissue and plasma levels of galectins in patients with high grade serous ovarian carcinoma as new predictive biomarkers

doi: 10.1038/s41598-017-13802-5

Figure Lengend Snippet: Combining gal-8 and gal-9 increases the predictive value for OS and HFS. ( A ) Kaplan-Meier curves of the 5-years OS and 5-years DFS according to the presence of either or both gal-8 and gal-9 in the plasma of HGSC patients. Blue bar: Gal-8 LOW /Gal-9 LOW , green bar: Gal-8 High /Gal-9 LOW , Yellow bar: Gal-8 LOW /Gal-9 High , Magenta bar: Gal-8 High /Gal-9 HIGH . ( B ) Correlation between the concentration of gal-8 and gal-9 in the plasma of HGSC patients. ( C ) Kaplan-Meier curves of the 5-years OS and 5-years DFS according to the presence of both gal-8 and gal-9 in the plasma of HGSC patients. Blue bar: Gal-8 LOW /Gal-9 LOW , Gal-8 High /Gal-9 LOW or Gal-8 LOW /Gal-9 High sample; Red bar: Gal-8 High /Gal-9 HIGH samples. Patients with plasma gal-8 ≥ 2.7 ng/ml or plasma gal-9 ≥ 0.73 ng/ml were considered Gal-8 High and Gal-9 High , respectively.

Article Snippet: Rabbit anti-human gal-9 (1:100, Abcam), goat anti-human gal-9 (1:50, R&D Systems), rabbit anti-human LC3B (1:200, Sigma) and rabbit anti-human Cox IV (1:500, New England Biolabs, Ipswich, MA) primary antibodies were used.

Techniques: Concentration Assay