lipid mediated gene delivery  (Thermo Fisher)


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    Structured Review

    Thermo Fisher lipid mediated gene delivery
    Lipid Mediated Gene Delivery, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 1 article reviews
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    Transfection:

    Article Title: Herpesvirus-Mediated Delivery of a Genetically Encoded Fluorescent Ca2+ Sensor to Canine Cardiomyocytes
    Article Snippet: .. Viral DNA was transfected to actively growing PK-15 cells along with the appropriate targeting plasmid using lipid-mediated gene delivery (Lipofectamine 2000 Reagent, Invitrogen). .. Viral DNA for the transfection was prepared from virions purified from the medium of infected cells showing total cytopathic effects by isopycnic centrifugation on a discontinuous gradient, as described previously [ ].

    Plasmid Preparation:

    Article Title: Herpesvirus-Mediated Delivery of a Genetically Encoded Fluorescent Ca2+ Sensor to Canine Cardiomyocytes
    Article Snippet: .. Viral DNA was transfected to actively growing PK-15 cells along with the appropriate targeting plasmid using lipid-mediated gene delivery (Lipofectamine 2000 Reagent, Invitrogen). .. Viral DNA for the transfection was prepared from virions purified from the medium of infected cells showing total cytopathic effects by isopycnic centrifugation on a discontinuous gradient, as described previously [ ].

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    Thermo Fisher gene exp scp2 mm01257982 m1
    <t>SCP2</t> deficiency significantly attenuates diet-induced atherosclerosis. At 10 weeks of age, LDLR −/− and LS mice were fed a high-fat, high-cholesterol–containing Western-type diet (TD88137) for 16 weeks. Aortae were dissected and prepared for en face analyses. The total area as well as the area occupied by the lesions was quantified using Axiovision software and expressed as percent lesion area. A , representative images. B , percent area occupied by the lesions in the aortic arch. C , percent area occupied by the lesions in the entire aorta. *, p
    Gene Exp Scp2 Mm01257982 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp scp2 mm01257982 m1/product/Thermo Fisher
    Average 87 stars, based on 4 article reviews
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    gene exp scp2 mm01257982 m1 - by Bioz Stars, 2020-09
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    92
    Thermo Fisher ega1 c1c2 proteins
    Decoration of RBC EVs with <t>EGa1-C1C2</t> dose-dependently increases EV binding and uptake specifically by EGFR-overexpressing A431 cells. A. Binding of AlexaFluor488-labeled RBC EVs decorated with increasing amounts of R2-C1C2 (gray) or EGa1-C1C2 (black) to Neuro2A cells (panel A) and A431 cells (panel B) for 1 hour at 4 °C, determined by flow cytometry. C. Uptake of AlexaFluor488-labeled RBC EVs decorated with increasing amounts of C1C2-nanobodies by Neuro2A cells (panel C) and A431 cells (panel D) for 4 hours at 37 °C, determined by flow cytometry. Representative data of at least 2 replicate experiments are shown and data are displayed as mean ± SD.
    Ega1 C1c2 Proteins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher di 8 anepps
    Characterization of the effects of phloretin, 6-ketocholestanol and atorvastatin on the biophysical properties of the membrane. Cells were pretreated with phloretin, 6-ketocholestanol or two different concentrations of atorvastatin (ATO) as described in Materials and methods. The dipole potential was measured with <t>di-8-ANEPPS,</t> whose excitation intensity ratio correlates positively with the dipole potential (A). The fluorescence anisotropy of TMA-DPH is inversely related to membrane fluidity (B), whereas the generalized polarization (GP) of Laurdan is proportional to the hydration or compactness of the membrane (C). The error bars display the standard error of the mean of six independent samples from two biological replicates. Asterisks indicate statistical significance (p
    Di 8 Anepps, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SCP2 deficiency significantly attenuates diet-induced atherosclerosis. At 10 weeks of age, LDLR −/− and LS mice were fed a high-fat, high-cholesterol–containing Western-type diet (TD88137) for 16 weeks. Aortae were dissected and prepared for en face analyses. The total area as well as the area occupied by the lesions was quantified using Axiovision software and expressed as percent lesion area. A , representative images. B , percent area occupied by the lesions in the aortic arch. C , percent area occupied by the lesions in the entire aorta. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

    doi: 10.1074/jbc.RA118.002290

    Figure Lengend Snippet: SCP2 deficiency significantly attenuates diet-induced atherosclerosis. At 10 weeks of age, LDLR −/− and LS mice were fed a high-fat, high-cholesterol–containing Western-type diet (TD88137) for 16 weeks. Aortae were dissected and prepared for en face analyses. The total area as well as the area occupied by the lesions was quantified using Axiovision software and expressed as percent lesion area. A , representative images. B , percent area occupied by the lesions in the aortic arch. C , percent area occupied by the lesions in the entire aorta. *, p

    Article Snippet: Total RNA from the liver or different segments of the gastrointestinal tract was prepared using RNeasy mini kits (Qiagen), and the mRNA levels of the indicated genes were determined using optimized TaqMan probe sets from Applied Biosystems: Abcg5, Mm00446249_m1; Abcg8, Mm00445970_m1; ApoB, Mm01545156_m1; Fas, Mm00662319_m1; Npc1l1, Mm01191973_m1; Scp-2, Mm01257982_m1; Srebp-1c, Mm00550338_m1; and Srebp-2, Mm01306292_m1.

    Techniques: Mouse Assay, Western Blot, Software

    SCP2 deficiency significantly reduces lipid secretion from liver and isolated hepatocytes. A , C57BL/6 (WT) or SCP2 −/− mice were injected with the lipoprotein lipase inhibitor tyloxapol (500 mg/kg of body weight), and blood samples were drawn at 0 and 3 h. Triglyceride secretion rates for indicated genotypes and sexes are presented. B and C , primary hepatocytes were prepared from C57BL/6 (WT) or SCP2 −/− mice. Following incubation with [ 3 H]oleic acid, radiolabel associated with secreted triglycerides ( TG , B ) or cholesteryl esters ( C ) was determined as described under “Experimental procedures” and normalized to cellular protein. Data are presented as DPM associated with the triglyceride or cholesteryl ester fraction in the total lipids extracted from the medium per milligram of total protein.

    Journal: The Journal of Biological Chemistry

    Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

    doi: 10.1074/jbc.RA118.002290

    Figure Lengend Snippet: SCP2 deficiency significantly reduces lipid secretion from liver and isolated hepatocytes. A , C57BL/6 (WT) or SCP2 −/− mice were injected with the lipoprotein lipase inhibitor tyloxapol (500 mg/kg of body weight), and blood samples were drawn at 0 and 3 h. Triglyceride secretion rates for indicated genotypes and sexes are presented. B and C , primary hepatocytes were prepared from C57BL/6 (WT) or SCP2 −/− mice. Following incubation with [ 3 H]oleic acid, radiolabel associated with secreted triglycerides ( TG , B ) or cholesteryl esters ( C ) was determined as described under “Experimental procedures” and normalized to cellular protein. Data are presented as DPM associated with the triglyceride or cholesteryl ester fraction in the total lipids extracted from the medium per milligram of total protein.

    Article Snippet: Total RNA from the liver or different segments of the gastrointestinal tract was prepared using RNeasy mini kits (Qiagen), and the mRNA levels of the indicated genes were determined using optimized TaqMan probe sets from Applied Biosystems: Abcg5, Mm00446249_m1; Abcg8, Mm00445970_m1; ApoB, Mm01545156_m1; Fas, Mm00662319_m1; Npc1l1, Mm01191973_m1; Scp-2, Mm01257982_m1; Srebp-1c, Mm00550338_m1; and Srebp-2, Mm01306292_m1.

    Techniques: Isolation, Mouse Assay, Injection, Incubation

    SCP2 deficiency significantly reduces plasma cholesterol and triglyceride levels. At 10 weeks of age, LDLR −/− and LS mice were fed a high-fat, high-cholesterol–containing Western-type diet (TD88137) for 16 weeks. After an overnight fast, mice were euthanized, and fasting plasma was collected. A , levels of total plasma cholesterol for indicated genotype and sexes. B , percent of total plasma cholesterol associated with the non-HDL or HDL fraction. C , percent of total plasma cholesterol associated with the non-HDL or HDL fraction in both genotypes normalized to total plasma cholesterol in LDLR −/− mice of the respective sex. D , linear regression analyses of total lesion area and plasma cholesterol; the observed coefficient of correlation (R) as significance of correlation ( p value) is indicated. E , total plasma triglyceride for the indicated genotype and sexes. F , linear regression analyses of total lesion area and plasma triglyceride levels; the observed coefficients of correlation (R) as significance of correlation ( p value) is indicated.

    Journal: The Journal of Biological Chemistry

    Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

    doi: 10.1074/jbc.RA118.002290

    Figure Lengend Snippet: SCP2 deficiency significantly reduces plasma cholesterol and triglyceride levels. At 10 weeks of age, LDLR −/− and LS mice were fed a high-fat, high-cholesterol–containing Western-type diet (TD88137) for 16 weeks. After an overnight fast, mice were euthanized, and fasting plasma was collected. A , levels of total plasma cholesterol for indicated genotype and sexes. B , percent of total plasma cholesterol associated with the non-HDL or HDL fraction. C , percent of total plasma cholesterol associated with the non-HDL or HDL fraction in both genotypes normalized to total plasma cholesterol in LDLR −/− mice of the respective sex. D , linear regression analyses of total lesion area and plasma cholesterol; the observed coefficient of correlation (R) as significance of correlation ( p value) is indicated. E , total plasma triglyceride for the indicated genotype and sexes. F , linear regression analyses of total lesion area and plasma triglyceride levels; the observed coefficients of correlation (R) as significance of correlation ( p value) is indicated.

    Article Snippet: Total RNA from the liver or different segments of the gastrointestinal tract was prepared using RNeasy mini kits (Qiagen), and the mRNA levels of the indicated genes were determined using optimized TaqMan probe sets from Applied Biosystems: Abcg5, Mm00446249_m1; Abcg8, Mm00445970_m1; ApoB, Mm01545156_m1; Fas, Mm00662319_m1; Npc1l1, Mm01191973_m1; Scp-2, Mm01257982_m1; Srebp-1c, Mm00550338_m1; and Srebp-2, Mm01306292_m1.

    Techniques: Mouse Assay, Western Blot

    SCP2 deficiency reduces plaque size as well as plaque necrosis in the aortic root. Paraffin-embedded aortic root sections (5 μm) were stained with H E or Masson's trichrome, imaged, and analyzed by Axiovision software. A , representative H E stained images of the aortic root of the indicated genotypes/sex. Scale bar = 100 μm. B , the total aortic root and area occupied by the lesions was quantified, and data (mean ± S.D., n = 6) are presented as percent lesion area. C , representative trichrome-stained images of the indicated genotypes/sex. Scale bar = 50 μm. D , the total and necrotic areas were quantified for all three aortic valve leaflets, and data (mean ± S.D., n = 12 leaflets) are presented as percent necrotic area. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

    doi: 10.1074/jbc.RA118.002290

    Figure Lengend Snippet: SCP2 deficiency reduces plaque size as well as plaque necrosis in the aortic root. Paraffin-embedded aortic root sections (5 μm) were stained with H E or Masson's trichrome, imaged, and analyzed by Axiovision software. A , representative H E stained images of the aortic root of the indicated genotypes/sex. Scale bar = 100 μm. B , the total aortic root and area occupied by the lesions was quantified, and data (mean ± S.D., n = 6) are presented as percent lesion area. C , representative trichrome-stained images of the indicated genotypes/sex. Scale bar = 50 μm. D , the total and necrotic areas were quantified for all three aortic valve leaflets, and data (mean ± S.D., n = 12 leaflets) are presented as percent necrotic area. *, p

    Article Snippet: Total RNA from the liver or different segments of the gastrointestinal tract was prepared using RNeasy mini kits (Qiagen), and the mRNA levels of the indicated genes were determined using optimized TaqMan probe sets from Applied Biosystems: Abcg5, Mm00446249_m1; Abcg8, Mm00445970_m1; ApoB, Mm01545156_m1; Fas, Mm00662319_m1; Npc1l1, Mm01191973_m1; Scp-2, Mm01257982_m1; Srebp-1c, Mm00550338_m1; and Srebp-2, Mm01306292_m1.

    Techniques: Staining, Software

    Effects of SCP2 deficiency on cholesterol accumulation in and cholesterol efflux from macrophages. Thioglycolate-elicited macrophages were isolated from C57BL/6 (WT) and SCP2 −/− mice and incubated with AcLDL (25 μg/ml) for 48 h. A and B , following two washes with PBS, cells were either fixed and stained with Oil Red O ( A ) or used for total lipid extraction and cholesterol mass measurement ( B ). Total cholesterol mass was normalized to total cellular protein, and data (mean ± S.D., n = 6) are presented as nanomoles per milligram of protein. C , total protein extracts of macrophages were subjected to Western blot analyses to assess SR-A expression; β-actin was used as a loading control. D , for measurement of cholesterol efflux, cells were loaded with AcLDL and labeled with [ 3 H]-cholesterol for 48 h. Following a 24-h equilibration, cholesterol efflux to 10% FBS in the growth medium was monitored over time. Data (mean ± S.D., n = 6) are presented as percent efflux. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

    doi: 10.1074/jbc.RA118.002290

    Figure Lengend Snippet: Effects of SCP2 deficiency on cholesterol accumulation in and cholesterol efflux from macrophages. Thioglycolate-elicited macrophages were isolated from C57BL/6 (WT) and SCP2 −/− mice and incubated with AcLDL (25 μg/ml) for 48 h. A and B , following two washes with PBS, cells were either fixed and stained with Oil Red O ( A ) or used for total lipid extraction and cholesterol mass measurement ( B ). Total cholesterol mass was normalized to total cellular protein, and data (mean ± S.D., n = 6) are presented as nanomoles per milligram of protein. C , total protein extracts of macrophages were subjected to Western blot analyses to assess SR-A expression; β-actin was used as a loading control. D , for measurement of cholesterol efflux, cells were loaded with AcLDL and labeled with [ 3 H]-cholesterol for 48 h. Following a 24-h equilibration, cholesterol efflux to 10% FBS in the growth medium was monitored over time. Data (mean ± S.D., n = 6) are presented as percent efflux. *, p

    Article Snippet: Total RNA from the liver or different segments of the gastrointestinal tract was prepared using RNeasy mini kits (Qiagen), and the mRNA levels of the indicated genes were determined using optimized TaqMan probe sets from Applied Biosystems: Abcg5, Mm00446249_m1; Abcg8, Mm00445970_m1; ApoB, Mm01545156_m1; Fas, Mm00662319_m1; Npc1l1, Mm01191973_m1; Scp-2, Mm01257982_m1; Srebp-1c, Mm00550338_m1; and Srebp-2, Mm01306292_m1.

    Techniques: Isolation, Mouse Assay, Incubation, Staining, Mass Measurement, Western Blot, Expressing, Labeling

    Effects of SCP2 deficiency on biliary bile acid and cholesterol secretion. A and B , gall bladder bile was collected at the time of euthanasia, and the total volume was noted. Biliary bile acids ( BA ), cholesterol, and phospholipids ( PL ) were estimated as described under “Experimental procedures.” Data are presented as total bile acids (nanomoles) or FC (micrograms) in the bile normalized to total phospholipids (micrograms) in A and B , respectively.

    Journal: The Journal of Biological Chemistry

    Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

    doi: 10.1074/jbc.RA118.002290

    Figure Lengend Snippet: Effects of SCP2 deficiency on biliary bile acid and cholesterol secretion. A and B , gall bladder bile was collected at the time of euthanasia, and the total volume was noted. Biliary bile acids ( BA ), cholesterol, and phospholipids ( PL ) were estimated as described under “Experimental procedures.” Data are presented as total bile acids (nanomoles) or FC (micrograms) in the bile normalized to total phospholipids (micrograms) in A and B , respectively.

    Article Snippet: Total RNA from the liver or different segments of the gastrointestinal tract was prepared using RNeasy mini kits (Qiagen), and the mRNA levels of the indicated genes were determined using optimized TaqMan probe sets from Applied Biosystems: Abcg5, Mm00446249_m1; Abcg8, Mm00445970_m1; ApoB, Mm01545156_m1; Fas, Mm00662319_m1; Npc1l1, Mm01191973_m1; Scp-2, Mm01257982_m1; Srebp-1c, Mm00550338_m1; and Srebp-2, Mm01306292_m1.

    Techniques:

    SCP2 deficiency leads to reduced lipid accumulation in the liver without a change in the expression of lipogenic genes. A , liver tissue harvested from WD-fed LDLR −/− or LS mice were paraffin-embedded, and 5-μm sections were stained with H E. Images were acquired using a Zeiss inverted microscope fitted with a digital camera. Scale bar = 50 μm. B, SCP2 mRNA levels in total liver RNA were determined by real-time PCR as described under “Experimental procedures” and normalized to the housekeeping gene β-actin. Relative expression (mean ± S.D., n = 6) is shown. C , hepatic mRNA levels of the indicated genes were determined by real-time PCR as described under “Experimental procedures” and normalized to the housekeeping gene β-actin. Relative expression (mean ± S.D., n = 6) is shown.

    Journal: The Journal of Biological Chemistry

    Article Title: Sterol carrier protein-2 deficiency attenuates diet-induced dyslipidemia and atherosclerosis in mice

    doi: 10.1074/jbc.RA118.002290

    Figure Lengend Snippet: SCP2 deficiency leads to reduced lipid accumulation in the liver without a change in the expression of lipogenic genes. A , liver tissue harvested from WD-fed LDLR −/− or LS mice were paraffin-embedded, and 5-μm sections were stained with H E. Images were acquired using a Zeiss inverted microscope fitted with a digital camera. Scale bar = 50 μm. B, SCP2 mRNA levels in total liver RNA were determined by real-time PCR as described under “Experimental procedures” and normalized to the housekeeping gene β-actin. Relative expression (mean ± S.D., n = 6) is shown. C , hepatic mRNA levels of the indicated genes were determined by real-time PCR as described under “Experimental procedures” and normalized to the housekeeping gene β-actin. Relative expression (mean ± S.D., n = 6) is shown.

    Article Snippet: Total RNA from the liver or different segments of the gastrointestinal tract was prepared using RNeasy mini kits (Qiagen), and the mRNA levels of the indicated genes were determined using optimized TaqMan probe sets from Applied Biosystems: Abcg5, Mm00446249_m1; Abcg8, Mm00445970_m1; ApoB, Mm01545156_m1; Fas, Mm00662319_m1; Npc1l1, Mm01191973_m1; Scp-2, Mm01257982_m1; Srebp-1c, Mm00550338_m1; and Srebp-2, Mm01306292_m1.

    Techniques: Expressing, Mouse Assay, Staining, Inverted Microscopy, Real-time Polymerase Chain Reaction

    Decoration of RBC EVs with EGa1-C1C2 dose-dependently increases EV binding and uptake specifically by EGFR-overexpressing A431 cells. A. Binding of AlexaFluor488-labeled RBC EVs decorated with increasing amounts of R2-C1C2 (gray) or EGa1-C1C2 (black) to Neuro2A cells (panel A) and A431 cells (panel B) for 1 hour at 4 °C, determined by flow cytometry. C. Uptake of AlexaFluor488-labeled RBC EVs decorated with increasing amounts of C1C2-nanobodies by Neuro2A cells (panel C) and A431 cells (panel D) for 4 hours at 37 °C, determined by flow cytometry. Representative data of at least 2 replicate experiments are shown and data are displayed as mean ± SD.

    Journal: Nanoscale

    Article Title: Recombinant phosphatidylserine-binding nanobodies for targeting of extracellular vesicles to tumor cells: a plug-and-play approach phosphatidylserine-binding nanobodies for targeting of extracellular vesicles to tumor cells: a plug-and-play approach †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7nr06966a

    doi: 10.1039/c7nr06966a

    Figure Lengend Snippet: Decoration of RBC EVs with EGa1-C1C2 dose-dependently increases EV binding and uptake specifically by EGFR-overexpressing A431 cells. A. Binding of AlexaFluor488-labeled RBC EVs decorated with increasing amounts of R2-C1C2 (gray) or EGa1-C1C2 (black) to Neuro2A cells (panel A) and A431 cells (panel B) for 1 hour at 4 °C, determined by flow cytometry. C. Uptake of AlexaFluor488-labeled RBC EVs decorated with increasing amounts of C1C2-nanobodies by Neuro2A cells (panel C) and A431 cells (panel D) for 4 hours at 37 °C, determined by flow cytometry. Representative data of at least 2 replicate experiments are shown and data are displayed as mean ± SD.

    Article Snippet: To generate cell lines stably expressing R2-C1C2 or EGa1-C1C2 proteins, HEK293 cells were transfected overnight with pcDNA3.1-R2-C1C2 or pcDNA3.1-EGa1-C1C2 using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions.

    Techniques: Binding Assay, Labeling, Flow Cytometry, Cytometry

    Decoration of Neuro2A EVs with EGa1-C1C2 dose-dependently promotes EV uptake by EGFR-overexpressing A431 cells and inhibits uptake by non-targeted cells. A. Western blot of Neuro2A EVs after decoration with increasing amounts of R2-C1C2 and EGa1-C1C2, or with 300 ng μg –1 EGa1 without C1C2 domains (second lane) and after SEC purification. C1C2-nanobodies were detected with anti-Myc antibodies and Alix and CD9 were used as EV marker proteins. In each lane 3 μg of protein was loaded. In the last two lanes 100 ng of R2-C1C2 and EGa1-C1C2 were loaded as references. B. Uptake of CellTracker Deep Red labeled, SEC purified Neuro2A EVs decorated with increasing concentrations of R2-C1C2 (gray) or EGa1-C1C2 (black) or 300 ng μg –1 of EGa1 without C1C2 domains by A431 cells or Neuro2A cells (panel C), as determined by flow cytometry. D. Uptake of CellTracker Deep Red labeled Neuro2A EVs decorated with increasing concentrations of R2-C1C2 or EGa1-C1C2 or 300 ng μg –1 of EGa1 without C1C2 domains by CellTracker Green labeled Neuro2A cells co-cultured with unstained A431 cells in a 2 : 1 ratio, as determined by flow cytometry. Data are expressed as the ratio of mean fluorescence intensity (MFI) of A431 cells with that of Neuro2A cells in the same well. Representative data of at least 3 replicate experiments are shown and data are displayed as mean ± SD.

    Journal: Nanoscale

    Article Title: Recombinant phosphatidylserine-binding nanobodies for targeting of extracellular vesicles to tumor cells: a plug-and-play approach phosphatidylserine-binding nanobodies for targeting of extracellular vesicles to tumor cells: a plug-and-play approach †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7nr06966a

    doi: 10.1039/c7nr06966a

    Figure Lengend Snippet: Decoration of Neuro2A EVs with EGa1-C1C2 dose-dependently promotes EV uptake by EGFR-overexpressing A431 cells and inhibits uptake by non-targeted cells. A. Western blot of Neuro2A EVs after decoration with increasing amounts of R2-C1C2 and EGa1-C1C2, or with 300 ng μg –1 EGa1 without C1C2 domains (second lane) and after SEC purification. C1C2-nanobodies were detected with anti-Myc antibodies and Alix and CD9 were used as EV marker proteins. In each lane 3 μg of protein was loaded. In the last two lanes 100 ng of R2-C1C2 and EGa1-C1C2 were loaded as references. B. Uptake of CellTracker Deep Red labeled, SEC purified Neuro2A EVs decorated with increasing concentrations of R2-C1C2 (gray) or EGa1-C1C2 (black) or 300 ng μg –1 of EGa1 without C1C2 domains by A431 cells or Neuro2A cells (panel C), as determined by flow cytometry. D. Uptake of CellTracker Deep Red labeled Neuro2A EVs decorated with increasing concentrations of R2-C1C2 or EGa1-C1C2 or 300 ng μg –1 of EGa1 without C1C2 domains by CellTracker Green labeled Neuro2A cells co-cultured with unstained A431 cells in a 2 : 1 ratio, as determined by flow cytometry. Data are expressed as the ratio of mean fluorescence intensity (MFI) of A431 cells with that of Neuro2A cells in the same well. Representative data of at least 3 replicate experiments are shown and data are displayed as mean ± SD.

    Article Snippet: To generate cell lines stably expressing R2-C1C2 or EGa1-C1C2 proteins, HEK293 cells were transfected overnight with pcDNA3.1-R2-C1C2 or pcDNA3.1-EGa1-C1C2 using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions.

    Techniques: Western Blot, Size-exclusion Chromatography, Purification, Marker, Labeling, Flow Cytometry, Cytometry, Cell Culture, Fluorescence

    C1C2-proteins are expressed and secreted by HEK293 cells and can be purified from conditioned medium. A. Western blot of HEK293 cell lysates after stable transfection with pcDNA3.1-R2-C1C2 (R2) or pcDNA3.1-EGa1-C1C2 (EGa1) vectors (10 μg protein per lane) and corresponding conditioned medium (45 μL medium per lane). Myc tags were used to detect C1C2-nanobody expression, and β-actin was included as a loading control. B. Western blot of R2-C1C2 and EGa1-C1C2 after purification from conditioned medium, stained with anti-Myc antibodies. C. SDS-PAGE of purified R2-C1C2 and EGa1-C1C2. Arrows indicate bands of BSA from the OSB (gray) and C1C2-nanobodies (black). Lanes in B and C contained 1 μg of protein.

    Journal: Nanoscale

    Article Title: Recombinant phosphatidylserine-binding nanobodies for targeting of extracellular vesicles to tumor cells: a plug-and-play approach phosphatidylserine-binding nanobodies for targeting of extracellular vesicles to tumor cells: a plug-and-play approach †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7nr06966a

    doi: 10.1039/c7nr06966a

    Figure Lengend Snippet: C1C2-proteins are expressed and secreted by HEK293 cells and can be purified from conditioned medium. A. Western blot of HEK293 cell lysates after stable transfection with pcDNA3.1-R2-C1C2 (R2) or pcDNA3.1-EGa1-C1C2 (EGa1) vectors (10 μg protein per lane) and corresponding conditioned medium (45 μL medium per lane). Myc tags were used to detect C1C2-nanobody expression, and β-actin was included as a loading control. B. Western blot of R2-C1C2 and EGa1-C1C2 after purification from conditioned medium, stained with anti-Myc antibodies. C. SDS-PAGE of purified R2-C1C2 and EGa1-C1C2. Arrows indicate bands of BSA from the OSB (gray) and C1C2-nanobodies (black). Lanes in B and C contained 1 μg of protein.

    Article Snippet: To generate cell lines stably expressing R2-C1C2 or EGa1-C1C2 proteins, HEK293 cells were transfected overnight with pcDNA3.1-R2-C1C2 or pcDNA3.1-EGa1-C1C2 using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions.

    Techniques: Purification, Western Blot, Stable Transfection, Expressing, Staining, SDS Page

    C1C2-nanobodies self-associate with RBC EVs in a dose-dependent manner without affecting EV size and integrity. A. Western blot of RBC EVs after decoration with increasing amounts of R2-C1C2 and EGa1-C1C2 and after SEC purification. C1C2-nanobodies were detected with anti-Myc antibodies and alpha-1-spectrin was used as a loading control for EVs. In each lane 20 μg of protein was loaded. Right two lanes show typical EV fractions after loading of high concentrations of R2-C1C2 or EGa1-C1C2 (corresponding with concentrations used in sample lanes 4 and 7) onto the SEC column. B. Representative size distribution of RBC EVs after decoration with C1C2-nanobodies (40 ng μg –1 EV), determined by Nanoparticle Tracking Analysis. Data is displayed as mean ± SD of 5 measurements. C. Transmission electron microscopy pictures of SEC-purified RBC EVs after decoration with R2-C1C2 or EGa1-C1C2 at a concentration of 40 ng μg –1 EV. Immunogold labeling was performed with anti-Myc antibodies and arrowheads indicate examples of membrane-associated gold. Scale bars represent 200 nm.

    Journal: Nanoscale

    Article Title: Recombinant phosphatidylserine-binding nanobodies for targeting of extracellular vesicles to tumor cells: a plug-and-play approach phosphatidylserine-binding nanobodies for targeting of extracellular vesicles to tumor cells: a plug-and-play approach †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7nr06966a

    doi: 10.1039/c7nr06966a

    Figure Lengend Snippet: C1C2-nanobodies self-associate with RBC EVs in a dose-dependent manner without affecting EV size and integrity. A. Western blot of RBC EVs after decoration with increasing amounts of R2-C1C2 and EGa1-C1C2 and after SEC purification. C1C2-nanobodies were detected with anti-Myc antibodies and alpha-1-spectrin was used as a loading control for EVs. In each lane 20 μg of protein was loaded. Right two lanes show typical EV fractions after loading of high concentrations of R2-C1C2 or EGa1-C1C2 (corresponding with concentrations used in sample lanes 4 and 7) onto the SEC column. B. Representative size distribution of RBC EVs after decoration with C1C2-nanobodies (40 ng μg –1 EV), determined by Nanoparticle Tracking Analysis. Data is displayed as mean ± SD of 5 measurements. C. Transmission electron microscopy pictures of SEC-purified RBC EVs after decoration with R2-C1C2 or EGa1-C1C2 at a concentration of 40 ng μg –1 EV. Immunogold labeling was performed with anti-Myc antibodies and arrowheads indicate examples of membrane-associated gold. Scale bars represent 200 nm.

    Article Snippet: To generate cell lines stably expressing R2-C1C2 or EGa1-C1C2 proteins, HEK293 cells were transfected overnight with pcDNA3.1-R2-C1C2 or pcDNA3.1-EGa1-C1C2 using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions.

    Techniques: Western Blot, Size-exclusion Chromatography, Purification, Transmission Assay, Electron Microscopy, Concentration Assay, Labeling

    Purified C1C2-nanobodies bind to PS and EGFR with high affinity and compete with binding of EGF. A. Protein–lipid overlay assay in which a PVDF membrane was spotted with 500 pmol of phosphatidylglycerol (PG), cholesterol (CHL), ganglioside GM3 (GM3), sphingomyelin (SM), egg- and soy-derived phosphatidylcholine (PC egg and PC soy , respectively), phosphatidylethanolamine (PE), and decreasing quantities of PS. Binding of R2-C1C2 and EGa1-C1C2 was detected with anti-Myc antibodies. B. ELISA showing binding of increasing concentrations of R2-C1C2 and EGa1-C1C2 to immobilized extracellular domains of EGFR. C1C2-nanobody binding was quantified using anti-Myc antibodies with peroxidase detection. C. Competition ELISA in which C1C2-nanobodies were mixed with 40 nM EGF-IRDye800 (EGF-IR) and incubated with plate-captured extracellular domains of EGFR. EGF-IR binding was analyzed using an Odyssey imager. All data are displayed as mean ± SD and are representative of at least two replicate experiments.

    Journal: Nanoscale

    Article Title: Recombinant phosphatidylserine-binding nanobodies for targeting of extracellular vesicles to tumor cells: a plug-and-play approach phosphatidylserine-binding nanobodies for targeting of extracellular vesicles to tumor cells: a plug-and-play approach †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7nr06966a

    doi: 10.1039/c7nr06966a

    Figure Lengend Snippet: Purified C1C2-nanobodies bind to PS and EGFR with high affinity and compete with binding of EGF. A. Protein–lipid overlay assay in which a PVDF membrane was spotted with 500 pmol of phosphatidylglycerol (PG), cholesterol (CHL), ganglioside GM3 (GM3), sphingomyelin (SM), egg- and soy-derived phosphatidylcholine (PC egg and PC soy , respectively), phosphatidylethanolamine (PE), and decreasing quantities of PS. Binding of R2-C1C2 and EGa1-C1C2 was detected with anti-Myc antibodies. B. ELISA showing binding of increasing concentrations of R2-C1C2 and EGa1-C1C2 to immobilized extracellular domains of EGFR. C1C2-nanobody binding was quantified using anti-Myc antibodies with peroxidase detection. C. Competition ELISA in which C1C2-nanobodies were mixed with 40 nM EGF-IRDye800 (EGF-IR) and incubated with plate-captured extracellular domains of EGFR. EGF-IR binding was analyzed using an Odyssey imager. All data are displayed as mean ± SD and are representative of at least two replicate experiments.

    Article Snippet: To generate cell lines stably expressing R2-C1C2 or EGa1-C1C2 proteins, HEK293 cells were transfected overnight with pcDNA3.1-R2-C1C2 or pcDNA3.1-EGa1-C1C2 using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions.

    Techniques: Purification, Binding Assay, Protein-lipid Overlay Assay (PLOA), Derivative Assay, Enzyme-linked Immunosorbent Assay, Incubation

    Characterization of the effects of phloretin, 6-ketocholestanol and atorvastatin on the biophysical properties of the membrane. Cells were pretreated with phloretin, 6-ketocholestanol or two different concentrations of atorvastatin (ATO) as described in Materials and methods. The dipole potential was measured with di-8-ANEPPS, whose excitation intensity ratio correlates positively with the dipole potential (A). The fluorescence anisotropy of TMA-DPH is inversely related to membrane fluidity (B), whereas the generalized polarization (GP) of Laurdan is proportional to the hydration or compactness of the membrane (C). The error bars display the standard error of the mean of six independent samples from two biological replicates. Asterisks indicate statistical significance (p

    Journal: bioRxiv

    Article Title: Statin-boosted cellular uptake of penetratin due to reduced membrane dipole potential

    doi: 10.1101/2020.08.04.236984

    Figure Lengend Snippet: Characterization of the effects of phloretin, 6-ketocholestanol and atorvastatin on the biophysical properties of the membrane. Cells were pretreated with phloretin, 6-ketocholestanol or two different concentrations of atorvastatin (ATO) as described in Materials and methods. The dipole potential was measured with di-8-ANEPPS, whose excitation intensity ratio correlates positively with the dipole potential (A). The fluorescence anisotropy of TMA-DPH is inversely related to membrane fluidity (B), whereas the generalized polarization (GP) of Laurdan is proportional to the hydration or compactness of the membrane (C). The error bars display the standard error of the mean of six independent samples from two biological replicates. Asterisks indicate statistical significance (p

    Article Snippet: Membrane dipole potential measurement with di-8-ANEPPSAfter treatment with atorvastatin, 6-ketocholestanol or phloretin cells were incubated with di-8-ANEPPS (Thermo Fisher, D3167) at a final concentration of 2 μM on ice for 20 minutes ( , , ).

    Techniques: Fluorescence