Journal:

Article Title: Sustained Phenotypic Correction in a Mouse Model of Hypoalphalipoproteinemia with a Helper-Dependent Adenovirus Vector

doi: 10.1038/sj.gt.3302819

Figure Lengend Snippet: Structures of adenoviral vectors used for co-injection

Article Snippet: For APOE3 immunoblot, goat anti-APOE antibody (1:4000, Calbiochem) or mouse monoclonal anti-human APOE3 (1:1000, SIGNET Lab) were used.

Techniques: Control

Journal:

Article Title: Sustained Phenotypic Correction in a Mouse Model of Hypoalphalipoproteinemia with a Helper-Dependent Adenovirus Vector

doi: 10.1038/sj.gt.3302819

Figure Lengend Snippet: Co-injection of FGAd vectors suppresses hAPOA1 expression mediated by HDAd-AI vector. (a) Plasma human APOA1 levels in APOA1−/− mice treated with Ad vectors. *p<0.05 vs. PBS, **p<0.01. (b) Plasma cholesterol levels in APOA1−/− mice after intravenous injection of Ad vectors. *p<0.05 vs. PBS, **p<0.01. (c) Immunoblot analysis of transgene expression. 0.1 μl of plasma collected from mice 8 weeks after treatment with various Ad vectors was separated by SDS-PAGE, and the presence of transgene products was detected by immunoblot. Upper panel: goat anti-human APOA1 antibody which does not cross react with mouse APOA1; middle panel: goat anti-apoE antibody; lower panel: mouse monoclonal antibody against human APOE3. Lane 1: Human plasma; lane 2: APOE−/− mouse; lane 3: APOE−/− mouse treated with HDAd-E3; lane 4: APOA1−/− mouse treated with FGAd-AI + HDAd-0; lane 5: APOE−/− mouse treated with HDAd-mouse APOE; lane 6: APOA1−/− mouse treated with HDAd-AI + HDAd-0; lane 7: APOA1−/− mouse treated with HDAd-AI and HDAd-E3; lane 8: APOA1−/− mouse treated with HDAd-AI + FGAd-E3. (d) Generation of anti-hAPOA1 antibodies after intravenous co-injection of various Ad vectors. *p<0.05 (vs. day 0).

Article Snippet: For APOE3 immunoblot, goat anti-APOE antibody (1:4000, Calbiochem) or mouse monoclonal anti-human APOE3 (1:1000, SIGNET Lab) were used.

Techniques: Injection, Expressing, Plasmid Preparation, Clinical Proteomics, Western Blot, SDS Page

Journal: Viruses

Article Title: HBV Pre-S1-Derived Myristoylated Peptide (Myr47): Identification of the Inhibitory Activity on the Cellular Uptake of Lipid Nanoparticles

doi: 10.3390/v13050929

Figure Lengend Snippet: Effect of Myr47 and its mutants on the ApoE3-LP interaction. ( A ) Effect of ApoE3 on the cellular LP uptake activity in the presence of Myr47 or its mutants. Hep G2 cells were incubated with DiD-LPs (5 μg/mL) in serum-free DMEM containing Myr47 or its mutants (500 nM) with/without ApoE3 (25 μg/mL) for 3 h at 37 °C, and then analyzed with flow cytometry. Data are shown as geometric means ± S.D. ( n = 3). ( B ) Effect of Myr47 or its mutants on the interaction of LPs and ApoE3 was evaluated with BLItz. Data are shown as means. ( n = 3).

Article Snippet: Anti-Human ApoE3 IgG (1 μM) was contacted with a protein A sensor chip (Fortebio) for 240 s. The sensor chip was washed with K-buffer (PBS containing 0.1% BSA) for 30 s. ApoE3 (2.5 μM) could bind with the antibody on the sensor chip for 120 s. The sensor chip was washed with K-buffer for 120 s. Meanwhile, mixture of LPs (500 μg/mL) and Myr47 or its mutants (50 μg/mL) was incubated at RT for 20 min and put in 4-μL well.

Techniques: Activity Assay, Incubation, Flow Cytometry

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: CLCF1 binds recombinant and serum ApoE. ( A ) Recombinant CLCF1 (200 ng) and ApoE2, 3 or 4 (250 ng) were incubated alone or in combination for 16 h. The samples were subjected to immunoprecipitation (IP) with anti-CLCF1 and protein G agarose. Immunoprecipitated proteins were analyzed by WB using mAbs specific for ApoE (upper panel) or CLCF1 (lower panel). Lanes “CLC 2 ng” and “ApoE3 20 ng”, show results of recombinant proteins directly subjected to SDS-PAGE and WB blot analysis. ( B ) CLCF1 and ApoE form a complex when co-expressed in HEK-293 cells. The cell culture medium from stable transfectants expressing the indicated proteins in combination with CRLF1 or from cells transfected with empty expression vector (lane D5) was subjected to immunoaffinity chromatography with rat-anti-human ApoE and anti-rat Ig Agarose. The eluates were analysed by WB using anti-ApoE or anti-CLC mAbs (upper panels). Supernatants of the stable transfectants were analyzed for the presence of ApoE or CLCF1 by WB (lower panels). ( C and D ) CLCF1 binds mouse and human serum ApoE. ( C ) Mouse serum samples isolated from WT or ApoE −/− mice were diluted 1:20, mixed with biotinylated mouse CLCF1 and the complexes immunoprecipitated with rabbit anti-mouse ApoE and anti-rabbit Ig agarose. The eluates were analysed for ApoE or CLCF1 by WB using anti-mouse ApoE or HRP-labelled streptavidin. The last two lanes show the signals obtained with 40 ng of biotinylated mouse CLCF1 or ApoE included as control. ( D ) Human serum samples were diluted 1:20, mixed with biotinylated human CLCF1 and the complexes immunoprecipitated with rat anti-ApoE and anti-rat Ig agarose. The eluates were analyzed for ApoE or CLCF1 by WB using anti-ApoE or HRP-labelled streptavidin. The last two lanes show the signals obtained with 40 ng of biotinylated human CLCF1 or ApoE included as control. Blots’ images where cropped to show relevant areas.

Article Snippet: ApoE2, 3 or 4 (0–100 nM) were mixed with CLCF1 (10 nM) in PBS 0.1% BSA for 1 h with rat IgG anti-ApoE (3 nM) (R&D systems, Cedarlane) and anti-rat IgG acceptor beads (10 μg/mL).

Techniques: Recombinant, Incubation, Immunoprecipitation, SDS Page, Cell Culture, Expressing, Transfection, Plasmid Preparation, Chromatography, Isolation

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: AlphaLISA proximity assay shows CLCF1-ApoE complex formation that is increased by the lipid DMPC. ( A ) ApoE2, 3 or 4 were mixed with CLCF1 (10 nM) in PBS 0.1% BSA for 1 h with IgG rat anti-ApoE (3 nM) and anti-IgG rat acceptor beads (10 μg/mL). Streptavidin donor beads (40 μg/mL) were further added for 1 h and 615 nm fluorescence signal assessed. ( B ) The indicated ApoE isotypes (30 nM) were mixed at 37 °C with increasing amount of DMPC for 1 h. ApoE-DMPC mix were further incubated with CLCF1 and subjected to AlpahLISA proximity assay. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.

Article Snippet: ApoE2, 3 or 4 (0–100 nM) were mixed with CLCF1 (10 nM) in PBS 0.1% BSA for 1 h with rat IgG anti-ApoE (3 nM) (R&D systems, Cedarlane) and anti-rat IgG acceptor beads (10 μg/mL).

Techniques: Proximity Assay, Fluorescence, Incubation

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: CLCF1 co-purifies or co-elutes with serum lipoprotein complexes. ( A ) WT mouse, ( B ) human or ( C ) ApoE −/− mouse sera were mixed with corresponding biotinylated CLCF1. The mixtures were fractionated by FPLC using a Superose 6 HR 10/ column in order to obtain 40 fractions. The fractions were analyzed for ApoE or CLCF1 by WB using anti-ApoE or HRP-labelled streptavidin. The right lanes show the signals obtained with 40 ng of ApoE or biotinylated CLCF1 included as control. Blots’ images where cropped to show relevant areas.

Article Snippet: ApoE2, 3 or 4 (0–100 nM) were mixed with CLCF1 (10 nM) in PBS 0.1% BSA for 1 h with rat IgG anti-ApoE (3 nM) (R&D systems, Cedarlane) and anti-rat IgG acceptor beads (10 μg/mL).

Techniques:

Journal: Journal of genetic syndrome & gene therapy

Article Title: Gene Therapy Targeting LDL Cholesterol but not HDL Cholesterol Induces Regression of Advanced Atherosclerosis in a Mouse Model of Familial Hypercholesterolemia

doi:

Figure Lengend Snippet: Structure of helper-dependent adenoviral vectors expressing APOE3. A. Schematic presentation of helper-dependent adenoviral vectors (HDAd) containing phosphoenolpyruvate carboxykinase (PEPCK) promoter on C4HSU backbone. L-ITR and R-ITR indicate left and right adenovirus (Ad) inverted terminal repeat sequence, respectively; Ψ, Ad packaging signal; PEPCKpr, PEPCK promoter; AI intron, human apolipoprotein AI intron, hGH polyA, human growth hormone polyadenylation signal; WPRE, Woodchuck hepatitis virus post-transcriptional regulatory element; LCR, human APOE gene liver control region. B. HDAd containing human APOE gene promoter and liver specific enhancer, LCR. In HDAd-E-E3, the fragment containing exon 2–4 was removed and replaced with APOE3 cDNA. HDAd-gE3 contains APOE3 5′ flanking region as well as all exons. C. HDAd containing human APOAI promoter and 3′ flanking region. In HDAd-AI-E3, the APOAI gene corresponding to exon 2 to exon 4 was removed and replaced with APOE3 cDNA. D. HDAd containing human APOE3 gene and LCR on pΔ21 backbone. HPRT, intron region of human genomic HPRT; C346, cosmid C346 human genomic stuffer sequence.

Article Snippet: Human apoE3 levels were quantified by an ELISA using polyclonal goat anti-apoE antibody (Chemicon, 1:1000) and apoE3 (Calbiochem) as standard [ 18 ].

Techniques: Expressing, Sequencing

Journal: Journal of genetic syndrome & gene therapy

Article Title: Gene Therapy Targeting LDL Cholesterol but not HDL Cholesterol Induces Regression of Advanced Atherosclerosis in a Mouse Model of Familial Hypercholesterolemia

doi:

Figure Lengend Snippet: Plasma cholesterol levels. Six to eight week old Apoe−/− mice received i.v. injection of 5 × 1012 v.p./kg of HDAd expressing apoE3 and plasma cholesterol was measured over the following 32 weeks. A. PEPCK expression cassette. *p<0.05 vs. PBS, **p<0.001 vs. PBS, †p<0.01 vs. HDAd-P-E3, ‡p<0.05 vs. HDAd-P-E3 and HDAd-PW-E3. All time points in the HDAd-PW-E3 and HDAd-PWL-E3 groups were significantly different from the PBS group after vector treatment (p<0.001), but not indicated. B. APOAI expression cassette. p<0.001 vs. PBS, group, except in the HDAd-AI-E3 group 1 week after treatment. There was no statistical significance among treatment groups. C. APOE expression cassette. Plasma cholesterol levels in all treatment groups were significantly lower than those in the PBS group (p<0.001) except 1 week after treatment in the HDAd-E-E3 and HDAd-ghE3 groups. *p<0.05 vs. HDAd-EW-E3. **p<0.01 vs. HDAd-EW-E3 and HDAd-gE3. D. Plasma cholesterol levels in mice treated with HDAd-gE3 on pΔ21 backbone. *p<0.05 vs. HDAd-gE3, **p<0.01 vs. HDAd-gE3. n=5/group.

Article Snippet: Human apoE3 levels were quantified by an ELISA using polyclonal goat anti-apoE antibody (Chemicon, 1:1000) and apoE3 (Calbiochem) as standard [ 18 ].

Techniques: Injection, Expressing, Plasmid Preparation

Journal: Journal of genetic syndrome & gene therapy

Article Title: Gene Therapy Targeting LDL Cholesterol but not HDL Cholesterol Induces Regression of Advanced Atherosclerosis in a Mouse Model of Familial Hypercholesterolemia

doi:

Figure Lengend Snippet: Human ApoE3 levels in Apoe−/− mice after treatment with HDAd. A. PEPCK expression cassette. *p<0.05 vs. HDAd-P-E3 (n=5/group). B. APOAI cassette. *p<0.05 vs. HDAd-AIW-E3 at 2 weeks, **p<0.01 vs. HDAd-AIW-E3 and HDAd-AIWL at 1 week. C. APOE expression cassette. *p<0.05 vs. HDAd-EW-E3 and HDAdgE3, **p<0.01 vs. HDAd-EW-E3 and HDAd-gE3, and †p<0.05 vs. HDAd-EW-E3. D. Plasma apoE3 levels in mice treated with HDAd-gE3-D21. *p<0.05 vs. HDAd-gE3, **p<0.01 vs. HDAd-gE3.

Article Snippet: Human apoE3 levels were quantified by an ELISA using polyclonal goat anti-apoE antibody (Chemicon, 1:1000) and apoE3 (Calbiochem) as standard [ 18 ].

Techniques: Expressing

Journal:

Article Title: Sustained Phenotypic Correction in a Mouse Model of Hypoalphalipoproteinemia with a Helper-Dependent Adenovirus Vector

doi: 10.1038/sj.gt.3302819

Figure Lengend Snippet: Structures of adenoviral vectors used for co-injection

Article Snippet: For APOE3 immunoblot, goat anti-APOE antibody (1:4000, Calbiochem) or mouse monoclonal anti-human APOE3 (1:1000, SIGNET Lab) were used.

Techniques: Control

Journal:

Article Title: Sustained Phenotypic Correction in a Mouse Model of Hypoalphalipoproteinemia with a Helper-Dependent Adenovirus Vector

doi: 10.1038/sj.gt.3302819

Figure Lengend Snippet: Co-injection of FGAd vectors suppresses hAPOA1 expression mediated by HDAd-AI vector. (a) Plasma human APOA1 levels in APOA1−/− mice treated with Ad vectors. *p<0.05 vs. PBS, **p<0.01. (b) Plasma cholesterol levels in APOA1−/− mice after intravenous injection of Ad vectors. *p<0.05 vs. PBS, **p<0.01. (c) Immunoblot analysis of transgene expression. 0.1 μl of plasma collected from mice 8 weeks after treatment with various Ad vectors was separated by SDS-PAGE, and the presence of transgene products was detected by immunoblot. Upper panel: goat anti-human APOA1 antibody which does not cross react with mouse APOA1; middle panel: goat anti-apoE antibody; lower panel: mouse monoclonal antibody against human APOE3. Lane 1: Human plasma; lane 2: APOE−/− mouse; lane 3: APOE−/− mouse treated with HDAd-E3; lane 4: APOA1−/− mouse treated with FGAd-AI + HDAd-0; lane 5: APOE−/− mouse treated with HDAd-mouse APOE; lane 6: APOA1−/− mouse treated with HDAd-AI + HDAd-0; lane 7: APOA1−/− mouse treated with HDAd-AI and HDAd-E3; lane 8: APOA1−/− mouse treated with HDAd-AI + FGAd-E3. (d) Generation of anti-hAPOA1 antibodies after intravenous co-injection of various Ad vectors. *p<0.05 (vs. day 0).

Article Snippet: For APOE3 immunoblot, goat anti-APOE antibody (1:4000, Calbiochem) or mouse monoclonal anti-human APOE3 (1:1000, SIGNET Lab) were used.

Techniques: Injection, Expressing, Plasmid Preparation, Clinical Proteomics, Western Blot, SDS Page

Journal: EMBO Molecular Medicine

Article Title: Apolipoprotein E promotes subretinal mononuclear phagocyte survival and chronic inflammation in age-related macular degeneration

doi: 10.15252/emmm.201404524

Figure Lengend Snippet: APOE inhibits subretinal MP clearance Representative image of a RPE flatmount 12 h after the subretinal injection (red marking) of 4 μl PBS with 12,000 CFSE-stained thioglycollate-elicited peritoneal cells that contain 70% macrophages (inset close-up view). Quantifications of CFSE + F4/80 + macrophages at different time points after subretinal injections of C57BL/6J and Cx3cr1 GFP / GFP CFSE + macrophages ( n = 5/per group (12 h) and n = 6/per group thereafter; Mann–Whitney U -test, C57BL/6J versus Cx3cr1 GFP / GFP : 1 day n = 20/group * P < 0.0001; 2 day n = 6/group * P = 0.0317). Representative image of TUNEL/Hoechst double-staining 12 h after subretinal injection of C57BL/6J CFSE + macrophages (experiment repeated three times, inset close-up view). Representative cytometry images of SSC-A/CFSE and CD11b/F4/80 gated analysis of eye cell suspensions prepared 24 h after the injection of Cx3cr1 GFP / GFP CFSE + macrophages and cytometric quantification of eye cell suspensions at 24 h after the injection of C57BL/6J and Cx3cr1 GFP / GFP macrophages into C57BL/6J ( n = 16–20/group; Mann–Whitney U -test, * P = 0.0024). Quantification of subretinal CFSE + cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE + magnetic-bead-sorted bone marrow-derived monocytes (Mo) from C57BL/6J and Cx3cr1 GFP / GFP mice into C57BL/6J mice ( n = 8–12/group; Mann–Whitney U -test, * P = 0.0006). Quantification of subretinal CFSE + cells on RPE and retinal flatmounts 24 h after subretinal injections of CFSE + CD11b FACS-sorted brain microglial cells from C57BL/6J and Cx3cr1 GFP / GFP mice into C57BL/6J mice ( n = 9–12/group; Mann–Whitney U -test, * P = 0.0087). Quantification of subretinal CFSE + F4/80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injections of CFSE + macrophages from C57BL/6J, Cx3cr1 GFP / GFP , Cx3cr1 GFP / GFP A poE −/− , and ApoE −/− mice into C57BL/6J mice ( n = 8–12/group; one-way ANOVA/Dunnett test of Cx3cr1 GFP / GFP versus any other group * P ≤ 0.0001; Mann–Whitney U -test, Cx3cr1 GFP / GFP versus Cx3cr1 GFP / GFP A poE −/− * P = 0.0006). Quantification of subretinal CFSE + F4/80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injections of C57BL/6J CFSE + macrophages into C57BL/6J and with exogenously added APOE3 at 1, 10, or 100 μg/ml calculated intraocular concentrations ( n = 6–7/group; one-way ANOVA/Dunnett test: C57BL/6J versus 10 μg * P = 0.0488; C57BL/6J versus 100 μg ‡ P = 0.006. Mann–Whitney U -test: C57BL/6J versus 10 μg * P = 0.0012; C57BL/6J versus 100 μg ‡ P = 0.0013). Data information: All primary cells were prepared from male mice; all recipient C57BL/6J mice were male. Mo: monocytes; MC: microglial cells; Mϕ: macrophages; SCC-A: side scatter detector A. Scale bars: 1 mm (A); 50 μm (C).

Article Snippet: In specific experiments, cells were stimulated with recombinant human CX3CL1 or APOE3 (5 μg/ml, Leinco Technologies), APOE3 (5 μg/ml) with polymyxin B (25 μg/ml, Calbiochem), heat-denatured APOE3 (5 μg/ml, 95°C, 90 min), rat anti-IgG isotype control (100 μg/ml, R&D), rat anti-mouse CD14 (100 μg/ml, R&D), mouse anti-mouse TLR2 (100 μg/ml, Invivogen), and POS prepared as previously described (Molday et al , ).

Techniques: Injection, Staining, MANN-WHITNEY, TUNEL Assay, Double Staining, Cytometry, Derivative Assay

Journal: EMBO Molecular Medicine

Article Title: Apolipoprotein E promotes subretinal mononuclear phagocyte survival and chronic inflammation in age-related macular degeneration

doi: 10.15252/emmm.201404524

Figure Lengend Snippet: APOE promotes subretinal macrophage survival via IL-6 Quantitative RT–PCR of FasL mRNA normalized with β-actin mRNA of 12-month-old C57BL/6 RPE/choroid plexus 3 h after subretinal injection of PBS (PBS; n = 36), IL-6 ( n = 17), and APOE3 ( n = 21); calculated intraocular concentrations: 5 ng/ml and 10 μg/ml, respectively; one-way ANOVA/Dunnett post hoc test PBS versus IL-6 * P = 0.038; t -test PBS versus IL-6 * P = 0.043. Mouse IL-6 ELISA of supernatants from C57BL/6J peritoneal macrophages incubated for 24 h in control medium, lipid-free APOE3 (5 μg/ml), APOE3 (5 μg/ml), and polymyxin B (25 μg/ml), heat-denatured APOE3 (dAPOE3, 5 μg/ml), APOE3 (5 μg/ml), and rat IgG1 isotype control (IgG, 100 μg/ml) or APOE3 (5 μg/ml) and rat anti-CD14 antibody (aCD14 Ab, 100 μg/ml) or APOE3 (5 μg/ml) and rat anti-TLR2 antibody (aTLR2 Ab, 100 μg/ml) ( n = 5–6/group; one-way ANOVA/Bonferroni multi-comparison tests: APOE3 versus CTL * P < 0.0001; dAPOE3 versus APOE3 # P < 0.0001; APOE3 IgG versus CTL P < 0.0001; APOE3 IgG versus APOE3 aCD14 Ab § P < 0.0001; APOE3 IgG versus APOE3 aTLR2 Ab ++ P <0.0001. Mann–Whitney U -test: APOE3 versus CTL * P = 0.0043; dAPOE3 versus APOE3 # P = 0.0117; APOE3 IgG versus CTL P = 0.0080; APOE3 IgG versus APOE3 aCD14 Ab § P = 0.0117; APOE3 IgG versus APOE3 aTLR2 Ab ++ P = 0.0079. The experiment was repeated twice with similar results). Quantitative RT–PCR of IL-6 mRNA normalized with S26 mRNA of C57BL/6J and Cx3cr1 GFP / GFP , and Cx3cr1 GFP / GFP ApoE −/− peritoneal macrophages cultured for 24 h with CX3CL1 ( n = 5 per group, one-way ANOVA/Bonferroni post hoc multi-comparison tests: C57BL/6J versus Cx3cr1 GFP / GFP * P < 0.0001 and Cx3cr1 GFP / GFP versus Cx3cr1 GFP /GFP ApoE −/− ‡ P < 0.0001. Mann–Whitney U -test: C57BL/6J versus Cx3cr1 GFP / GFP * P = 0.0079 and Cx3cr1 GFP / GFP versus Cx3cr1 GFP / GFP ApoE −/− ‡ P = 0.0079. The experiment was repeated twice with similar results.). Orthogonal and lateral Z-stack projection of IL-6 (D, red), IBA-1 + (E, green), and phalloidin (E, blue) of IBA + mononuclear phagocytes adjacent to the phalloidin + RPE of a retinal flatmount from a 12-month-old Cx3cr1 GFP / GFP mouse. (Representative of three independent experiments, immunostainings omitting the primary antibody served as negative controls). Quantification of subretinal CFSE + F4/80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injection of C57BL/6J CFSE + macrophages into C57BL/6J with or without IL-6 (n = 15-20/group; calculated intraocular concentrations of 5 ng/ml. Mann–Whitney U -test: * P < 0.0001). Quantification of subretinal CFSE + F4/80 + macrophages on RPE and retinal flatmounts 24 h after subretinal injection of Cx3cr1 GFP / GFP CFSE + Mϕs into C57BL/6J with control (IgG, n = 16) or anti-IL-6 antibody (aIL-6 Ab, n = 8; calculated intraocular concentrations 5 μg/ml; per group. Mann–Whitney U -test: * P = 0.0036). Graphical summary. 7 day laser-injured IBA-1 (green) and CD102 (red) double-stained RPE flatmounts of control IgG- (I) and anti-IL-6-treated (J) Cx3cr1 GFP / GFP mice. Quantification of subretinal IBA-1 + mononuclear phagocytes/impact localized on the lesion surrounding RPE of Cx3cr1 GFP / GFP mice treated with control IgG, IL-6-, or CD14-blocking antibodies (calculated intraocular concentration 5 μg/ml; n = 13–14/group, one-way ANOVA/Dunnett's post hoc tests of IgG versus any other group * P < 0.001. Mann–Whitney U -test IgG versus anti-IL-6 * P = 0.0021; IgG versus anti CD14 * P = 0.0028). Data information: All primary cells were prepared from male mice; all recipient C57BL/6J mice were male; ONL: outer nuclear layer; OS: outer segments; RPE: retinal pigment epithelium. Scale bars: 20 μm (D, E); 50 μm (I, J).

Article Snippet: In specific experiments, cells were stimulated with recombinant human CX3CL1 or APOE3 (5 μg/ml, Leinco Technologies), APOE3 (5 μg/ml) with polymyxin B (25 μg/ml, Calbiochem), heat-denatured APOE3 (5 μg/ml, 95°C, 90 min), rat anti-IgG isotype control (100 μg/ml, R&D), rat anti-mouse CD14 (100 μg/ml, R&D), mouse anti-mouse TLR2 (100 μg/ml, Invivogen), and POS prepared as previously described (Molday et al , ).

Techniques: Quantitative RT-PCR, Injection, Enzyme-linked Immunosorbent Assay, Incubation, Control, Comparison, MANN-WHITNEY, Cell Culture, Staining, Blocking Assay, Concentration Assay