mertk  (R&D Systems)

Journal: Frontiers in Pharmacology

Article Title: Pomegranate Peel Extract Decreases Plaque Necrosis and Advanced Atherosclerosis Progression in Apoe -/- Mice

doi: 10.3389/fphar.2022.888300

Figure Lengend Snippet: (A) TUNEL assay was conducted on aortic root sections from male and female mice treated with PPE or vehicle to determine the number of dead cells in the atherosclerotic plaque. Bar, 50 μm (B) In-situ efferocytosis assay was conducted by immunostaining macrophages with anti-F4/80 antibody (red) and dead cells using TUNEL reagent (green). The number of TUNEL + nuclei that were either associated with an F4/80 + macrophage (white arrow) or were lying free (white arrowheads) was quantified. The associated: free ratio was used as a measure of lesional macrophage efferocytosis efficiency. Bar, 10 µm (C) Immunostaining of aortic root sections with anti-F4/80 and anti-Mertk antibody. The mean fluorescence intensity of the staining was quantified in ImageJ by drawing an ROI around the F4/80 + intimal region of the plaque. Bar, 50 µm. Non-parametric Mann-Whitney U test was conducted to determine statistical significance. *, p < 0.05; **, p < 0.01.

Article Snippet: Biotinylated anti-Mertk antibody (BAF591, R&D Systems) was used as a detection antibody followed by incubation with streptavidin-HRP and colorimetric development using TMB substrate.

Techniques: TUNEL Assay, In Situ, Immunostaining, Fluorescence, Staining, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: Pomegranate Peel Extract Decreases Plaque Necrosis and Advanced Atherosclerosis Progression in Apoe -/- Mice

doi: 10.3389/fphar.2022.888300

Figure Lengend Snippet: (A) qPCR-based analysis of Mertk mRNA expression in BMDMs treated with vehicle or 50 μg/ml PPE for 16 h n = 3 (B) Flow-cytometry based analysis of cell surface Mertk expression on BMDMs incubated with 50 μg/ml PPE for 16 h followed by treatment with 50 nM PMA for 2 h. The bar graph represents means fluorescence intensity derived from the histogram. n = 4 (C) The levels of soluble Mertk (Sol-Mertk) in the supernatants of BMDMs treated with PMA in the absence or presence of PPE (50 μg/ml) were analyzed by a sandwich ELISA. The bar graph represents the absorbance values measured at 450 nm n = 4 (D) Efferocytosis assay was conducted by incubating fluorescently labeled apoptotic cells (green) with BMDMs treated with PMA (50 nM) in the absence or presence of PPE (50 μg/ml). Bar, 20 μm n = 4 (E) Fluorescence microscopy-based analysis of cytosolic ROS by CellROX staining in BMDMs incubated with vehicle or PPE (50 μg/ml) for 16 h followed by treatment with 50 nM PMA for 2 h. Bar, 20 μm n = 4 (F) Aortic root sections of control or PPE-fed male and female Apoe -/- mice ( n = 5 mice per group per sex) were immunostained with anti-8-OHdG to assay lesional ROS-mediated damage. The white dotted line demarcates the intimal region of the plaque. Data were tested for statistical significance using a two-tailed Student’s t-test (A) , one-way ANOVA with Sidak’s multiple comparisons test (B–E) , or Mann–Whitney test (F) . ns, no significant difference; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: Biotinylated anti-Mertk antibody (BAF591, R&D Systems) was used as a detection antibody followed by incubation with streptavidin-HRP and colorimetric development using TMB substrate.

Techniques: Expressing, Flow Cytometry, Incubation, Fluorescence, Derivative Assay, Sandwich ELISA, Labeling, Microscopy, Staining, Control, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: Pomegranate Peel Extract Decreases Plaque Necrosis and Advanced Atherosclerosis Progression in Apoe -/- Mice

doi: 10.3389/fphar.2022.888300

Figure Lengend Snippet: (A) TUNEL assay was conducted on aortic root sections from male and female mice treated with PPE or vehicle to determine the number of dead cells in the atherosclerotic plaque. Bar, 50 μm (B) In-situ efferocytosis assay was conducted by immunostaining macrophages with anti-F4/80 antibody (red) and dead cells using TUNEL reagent (green). The number of TUNEL + nuclei that were either associated with an F4/80 + macrophage (white arrow) or were lying free (white arrowheads) was quantified. The associated: free ratio was used as a measure of lesional macrophage efferocytosis efficiency. Bar, 10 µm (C) Immunostaining of aortic root sections with anti-F4/80 and anti-Mertk antibody. The mean fluorescence intensity of the staining was quantified in ImageJ by drawing an ROI around the F4/80 + intimal region of the plaque. Bar, 50 µm. Non-parametric Mann-Whitney U test was conducted to determine statistical significance. *, p < 0.05; **, p < 0.01.

Article Snippet: The sections were incubated with appropriate primary antibodies: Biotinylated anti-mouse F4/80 (13-4801-82, 1:100, Invitrogen), anti-mouse F4/80 (14-4801-82, 1:100 dilutions, Invitrogen) biotinylated anti-mouse CD3 (36-0031-85, 1:50 dilution, Invitrogen), anti-smooth muscle actin (A2547, 1:100 dilution, Sigma), anti-mouse Mertk (BAF591, 1:100 dilution, R&D Systems), or anti-8-OHdG (BS-1278R, 1:100 dilution, Thermofisher Scientific) for 16 h at 4°C.

Techniques: TUNEL Assay, In Situ, Immunostaining, Fluorescence, Staining, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: Pomegranate Peel Extract Decreases Plaque Necrosis and Advanced Atherosclerosis Progression in Apoe -/- Mice

doi: 10.3389/fphar.2022.888300

Figure Lengend Snippet: (A) qPCR-based analysis of Mertk mRNA expression in BMDMs treated with vehicle or 50 μg/ml PPE for 16 h n = 3 (B) Flow-cytometry based analysis of cell surface Mertk expression on BMDMs incubated with 50 μg/ml PPE for 16 h followed by treatment with 50 nM PMA for 2 h. The bar graph represents means fluorescence intensity derived from the histogram. n = 4 (C) The levels of soluble Mertk (Sol-Mertk) in the supernatants of BMDMs treated with PMA in the absence or presence of PPE (50 μg/ml) were analyzed by a sandwich ELISA. The bar graph represents the absorbance values measured at 450 nm n = 4 (D) Efferocytosis assay was conducted by incubating fluorescently labeled apoptotic cells (green) with BMDMs treated with PMA (50 nM) in the absence or presence of PPE (50 μg/ml). Bar, 20 μm n = 4 (E) Fluorescence microscopy-based analysis of cytosolic ROS by CellROX staining in BMDMs incubated with vehicle or PPE (50 μg/ml) for 16 h followed by treatment with 50 nM PMA for 2 h. Bar, 20 μm n = 4 (F) Aortic root sections of control or PPE-fed male and female Apoe -/- mice ( n = 5 mice per group per sex) were immunostained with anti-8-OHdG to assay lesional ROS-mediated damage. The white dotted line demarcates the intimal region of the plaque. Data were tested for statistical significance using a two-tailed Student’s t-test (A) , one-way ANOVA with Sidak’s multiple comparisons test (B–E) , or Mann–Whitney test (F) . ns, no significant difference; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The sections were incubated with appropriate primary antibodies: Biotinylated anti-mouse F4/80 (13-4801-82, 1:100, Invitrogen), anti-mouse F4/80 (14-4801-82, 1:100 dilutions, Invitrogen) biotinylated anti-mouse CD3 (36-0031-85, 1:50 dilution, Invitrogen), anti-smooth muscle actin (A2547, 1:100 dilution, Sigma), anti-mouse Mertk (BAF591, 1:100 dilution, R&D Systems), or anti-8-OHdG (BS-1278R, 1:100 dilution, Thermofisher Scientific) for 16 h at 4°C.

Techniques: Expressing, Flow Cytometry, Incubation, Fluorescence, Derivative Assay, Sandwich ELISA, Labeling, Microscopy, Staining, Control, Two Tailed Test, MANN-WHITNEY