sheep anti cd39 entpd1  (R&D Systems)

Journal: Journal of Lipid Research

Article Title: Complete deletion of Cd39 is atheroprotective in apolipoprotein E-deficient mice [S]

doi: 10.1194/jlr.M072132

Figure Lengend Snippet: Cd39 deletion attenuates the formation of atherosclerotic lesions in ApoE -deficient mice. Male ApoE KO and DKO mice were fed a chow diet (n = 9 and 7 mice, respectively) (A) or a Western diet (n = 12 and 8 mice, respectively) (C) for up to 20 weeks and atherosclerotic lesion sizes were determined by Oil Red O staining of the whole aorta. Atherosclerotic lesions were also evaluated by Elastica van Gieson staining of the aortic root of ApoE KO and DKO mice fed a chow diet (n = 8 and 10 mice, respectively) (B) or a Western Diet (n = 7 mice in both groups) (D). Intimal plaque area was measured in four 50 μm distant sections per each animal. Data are presented as mean ± SEM. Representative images are shown. Scale bar in (B) and (D) is 250 μm. * P < 0.05; ** P < 0.01.

Article Snippet: The antibodies used were: rat anti-mouse Mac3 (M3/84, #550292, 1:150; BD Pharmingen); mouse anti-human Smc α actin (1A4, #M0851, 1:150; Dako); rabbit anti-human Cd3g (EPR4517, #ab134096, 1:100; Abcam); rabbit anti-mouse cleaved caspase 3 (5A1E, #9664, 1:100; Cell Signaling); and polyclonal sheep anti-mouse Cd39 (#AF4398; 1:500; R&D Systems).

Techniques: Western Blot, Staining

Journal: Journal of Lipid Research

Article Title: Complete deletion of Cd39 is atheroprotective in apolipoprotein E-deficient mice [S]

doi: 10.1194/jlr.M072132

Figure Lengend Snippet: Cd39 deletion results in higher HDL levels in serum from Western diet-fed mice. Peripheral blood from Western diet-fed ApoE KO and DKO mice was analyzed for levels of total cholesterol (A), LDL-C (B), and HDL-C (C) (n = 4–5 mice per group). Data are presented as mean ± SEM. ** P < 0.01.

Article Snippet: The antibodies used were: rat anti-mouse Mac3 (M3/84, #550292, 1:150; BD Pharmingen); mouse anti-human Smc α actin (1A4, #M0851, 1:150; Dako); rabbit anti-human Cd3g (EPR4517, #ab134096, 1:100; Abcam); rabbit anti-mouse cleaved caspase 3 (5A1E, #9664, 1:100; Cell Signaling); and polyclonal sheep anti-mouse Cd39 (#AF4398; 1:500; R&D Systems).

Techniques: Western Blot

Journal: Journal of Lipid Research

Article Title: Complete deletion of Cd39 is atheroprotective in apolipoprotein E-deficient mice [S]

doi: 10.1194/jlr.M072132

Figure Lengend Snippet: Cd39 deletion results in decreased macrophage numbers in fatty streaks from chow diet-fed mice. A: Macrophage levels were evaluated by Mac3 immunohistochemistry on aortic sinuses (one section per animal; n = 8 ApoE KO and 10 DKO mice). B: VSMCs infiltration was evaluated by Smc α actin immunohistochemistry on aortic sinus (one section per animal; n = 8 ApoE KO and 9 DKO mice). C: Intra-lesion apoptosis was investigated by TUNEL assay on aortic sinus (one section per animal; n = 7 ApoE KO and 10 DKO mice). Representative images are shown (scale bar is 250 μm in (A) and (B), 100 μm in (C). Quantification data are presented as mean ± SEM of the area staining or the percentage staining of the total plaque area. Setting a color threshold in ImageJ identified positive staining. * P < 0.05.

Article Snippet: The antibodies used were: rat anti-mouse Mac3 (M3/84, #550292, 1:150; BD Pharmingen); mouse anti-human Smc α actin (1A4, #M0851, 1:150; Dako); rabbit anti-human Cd3g (EPR4517, #ab134096, 1:100; Abcam); rabbit anti-mouse cleaved caspase 3 (5A1E, #9664, 1:100; Cell Signaling); and polyclonal sheep anti-mouse Cd39 (#AF4398; 1:500; R&D Systems).

Techniques: Immunohistochemistry, TUNEL Assay, Staining

Journal: Journal of Lipid Research

Article Title: Complete deletion of Cd39 is atheroprotective in apolipoprotein E-deficient mice [S]

doi: 10.1194/jlr.M072132

Figure Lengend Snippet: Cd39 deletion results in less necrosis in advanced lesions from Western diet-fed mice. A: Acellular necrotic area was measured by hematoxylin and eosin staining on aortic sinus. B: Intra-lesion apoptosis was evaluated by TUNEL assay on aortic sinus. C: Intra-lesion collagen content was quantified by Sirius red staining on aortic sinus. Necrotic and collagen-rich fibrotic areas were measured in four 50 μm distant sections per each animal, while the TUNEL-positive area was measured in one section per animal (n = 8 ApoE KO and 7 DKO mice for all the analyses). Representative images are shown (scale bar is 250 μm in (A) and (C), 50 μm in (B). All quantification data are presented as mean ± SEM of the percentage staining of the total plaque area. Setting a color threshold in ImageJ identified positive staining. ** P < 0.01.

Article Snippet: The antibodies used were: rat anti-mouse Mac3 (M3/84, #550292, 1:150; BD Pharmingen); mouse anti-human Smc α actin (1A4, #M0851, 1:150; Dako); rabbit anti-human Cd3g (EPR4517, #ab134096, 1:100; Abcam); rabbit anti-mouse cleaved caspase 3 (5A1E, #9664, 1:100; Cell Signaling); and polyclonal sheep anti-mouse Cd39 (#AF4398; 1:500; R&D Systems).

Techniques: Western Blot, Staining, TUNEL Assay

Journal: Journal of Lipid Research

Article Title: Complete deletion of Cd39 is atheroprotective in apolipoprotein E-deficient mice [S]

doi: 10.1194/jlr.M072132

Figure Lengend Snippet: Cd39 deletion impairs platelet activation. A: In vitro aggregometry response of platelets from ApoE KO and DKO mice fed the standard chow diet following 2.5 μM ADP stimulation. Data are presented as percent of light transmission over the time of incubation. Trace 1 (blue): platelets from ApoE KO mice; trace 2 (black): platelets from DKO mice. Aggregometry response is representative of three ApoE KO and four DKO mice. B: In vivo platelet turnover in ApoE KO and DKO mice fed the standard chow diet. Platelets were labeled and then counted every 24 h for 4 days, as described in the Materials and Methods. Data are presented as mean ± SEM of the percentage of labeled platelets to total platelets over the time of analysis (n = 5 ApoE KO and 4 DKO mice). ** P < 0.01, *** P < 0.001.

Article Snippet: The antibodies used were: rat anti-mouse Mac3 (M3/84, #550292, 1:150; BD Pharmingen); mouse anti-human Smc α actin (1A4, #M0851, 1:150; Dako); rabbit anti-human Cd3g (EPR4517, #ab134096, 1:100; Abcam); rabbit anti-mouse cleaved caspase 3 (5A1E, #9664, 1:100; Cell Signaling); and polyclonal sheep anti-mouse Cd39 (#AF4398; 1:500; R&D Systems).

Techniques: Activation Assay, In Vitro, Transmission Assay, Incubation, In Vivo, Labeling

Journal: Journal of Lipid Research

Article Title: Complete deletion of Cd39 is atheroprotective in apolipoprotein E-deficient mice [S]

doi: 10.1194/jlr.M072132

Figure Lengend Snippet: Cd39 deficiency does not impact foam cell formation and viability of oxLDL-treated macrophages. A: ApoE KO and DKO BMDMs were incubated with 25, 100, or 200 μg/ml human oxLDL for 24 h in complete medium. Foam cell formation was evaluated by Oil Red O staining and Oil Red O quantification. Control: unstained cells. Representative images are shown (n = 3). B: ApoE KO and DKO BMDMs were incubated with 25, 100, or 200 μg/ml human oxLDL for 24 h in medium without M-csf. Viability was measured by Cell Counting Kit-8 assay. M-csf: control cells incubated in complete medium. w/o M-csf: control cells incubated in medium without M-csf (n = 4–8). All data are presented as mean ± SEM.

Article Snippet: The antibodies used were: rat anti-mouse Mac3 (M3/84, #550292, 1:150; BD Pharmingen); mouse anti-human Smc α actin (1A4, #M0851, 1:150; Dako); rabbit anti-human Cd3g (EPR4517, #ab134096, 1:100; Abcam); rabbit anti-mouse cleaved caspase 3 (5A1E, #9664, 1:100; Cell Signaling); and polyclonal sheep anti-mouse Cd39 (#AF4398; 1:500; R&D Systems).

Techniques: Incubation, Staining, Control, Cell Counting

Journal: Journal of Lipid Research

Article Title: Complete deletion of Cd39 is atheroprotective in apolipoprotein E-deficient mice [S]

doi: 10.1194/jlr.M072132

Figure Lengend Snippet: Cd39 deletion results in increased macrophage Abca1-mediated cholesterol efflux. A: Cholesterol efflux to ApoA1 (10 μg/ml) was measured in ApoE KO and DKO BMDMs. Data are expressed as final efflux after subtracting the background (blank) to ApoA1 efflux (n = 2). B: Cholesterol efflux from DKO BMDMs was measured in the presence of ApoA1 or ApoA1 plus apyrase (25 U/ml) (n = 3). C: Cholesterol efflux from ApoE KO BMDMs was measured in the presence of ApoA1 or ApoA1 plus POM1 (26 μM) (n = 3). Blank: control with no acceptor (ApoA-1). All data are presented as mean ± SEM. * P < 0.05. ** P < 0.01.

Article Snippet: The antibodies used were: rat anti-mouse Mac3 (M3/84, #550292, 1:150; BD Pharmingen); mouse anti-human Smc α actin (1A4, #M0851, 1:150; Dako); rabbit anti-human Cd3g (EPR4517, #ab134096, 1:100; Abcam); rabbit anti-mouse cleaved caspase 3 (5A1E, #9664, 1:100; Cell Signaling); and polyclonal sheep anti-mouse Cd39 (#AF4398; 1:500; R&D Systems).

Techniques: Control

Journal: Journal of Lipid Research

Article Title: Complete deletion of Cd39 is atheroprotective in apolipoprotein E-deficient mice [S]

doi: 10.1194/jlr.M072132

Figure Lengend Snippet: Cd39 deletion in BM-derived cells exacerbates atherosclerosis and provokes platelet activation. Recipient ApoE KO and DKO mice were lethally irradiated and then transplanted with BM cells from either ApoE KO (wt) or DKO (KO) donors. Mice were fed a Western diet for 12 weeks. A: Atherosclerosis lesion size was evaluated by Oil Red O staining of the whole aorta (n = 8 wt → wt, 9 wt → KO, 8 KO → wt, and 7 KO → KO mice). Representative images are shown. B: HDL-C concentration was measured in plasma from transplanted mice (n ≥ 5 mice per group). C: Pf4 concentration in plasma from transplanted mice was determined by ELISA (n = 7 wt → wt, 9 wt → KO, 7 KO → wt, and 5 KO → KO mice). All data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. D: In vitro aggregometry response of platelets from BM-transplanted mice fed a standard Chow diet following 2.5 μM ADP stimulation. Data are presented as percent of light transmission over the time of incubation. Trace 1 (blue), platelets from wt → wt mice; trace 2 (black), platelets from wt → KO mice; trace 3 (red), platelets from KO → wt mice; and trace 4 (green), platelets from DKO mice. Aggregometry responses are representative of three mice per group.

Article Snippet: The antibodies used were: rat anti-mouse Mac3 (M3/84, #550292, 1:150; BD Pharmingen); mouse anti-human Smc α actin (1A4, #M0851, 1:150; Dako); rabbit anti-human Cd3g (EPR4517, #ab134096, 1:100; Abcam); rabbit anti-mouse cleaved caspase 3 (5A1E, #9664, 1:100; Cell Signaling); and polyclonal sheep anti-mouse Cd39 (#AF4398; 1:500; R&D Systems).

Techniques: Derivative Assay, Activation Assay, Irradiation, Western Blot, Staining, Concentration Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, In Vitro, Transmission Assay, Incubation