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Mark A Borden - One of the best experts on this subject based on the ideXlab platform.
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microbubble size isolation by Differential Centrifugation
Journal of Colloid and Interface Science, 2009Co-Authors: Jameel A Feshitan, Cherry C Chen, James J Kwan, Mark A BordenAbstract:Abstract Microbubbles used as contrast agents for ultrasound imaging, vectors for targeted drug delivery and vehicles for metabolic gas transport require better size control for improved performance. Mechanical agitation is the only method currently available to produce microbubbles in sufficient yields for biomedical applications, but the emulsions tend to be polydisperse. Herein, we describe a study to generate lipid-coated, perfluorobutane-filled microbubbles and isolate their size fractions based on migration in a centrifugal field. Polydispersity of the freshly sonicated suspension was characterized by particle sizing and counting through light obscuration/scattering and electrical impedance sensing, fluorescence and bright-field microscopy and flow cytometry. We found that the size distribution was multimodal. Smaller microbubbles were more abundant. Differential Centrifugation was used to successfully isolate the 1–2 and 4–5 μm diameter fractions. Isolated microbubbles were stable over two days. After two weeks, however, more dilute suspensions (
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microbubble size isolation by Differential Centrifugation
Journal of Colloid and Interface Science, 2009Co-Authors: Jameel A Feshitan, Cherry C Chen, James J Kwan, Mark A BordenAbstract:Microbubbles used as contrast agents for ultrasound imaging, vectors for targeted drug delivery and vehicles for metabolic gas transport require better size control for improved performance. Mechanical agitation is the only method currently available to produce microbubbles in sufficient yields for biomedical applications, but the emulsions tend to be polydisperse. Herein, we describe a study to generate lipid-coated, perfluorobutane-filled microbubbles and isolate their size fractions based on migration in a centrifugal field. Polydispersity of the freshly sonicated suspension was characterized by particle sizing and counting through light obscuration/scattering and electrical impedance sensing, fluorescence and bright-field microscopy and flow cytometry. We found that the size distribution was multimodal. Smaller microbubbles were more abundant. Differential Centrifugation was used to successfully isolate the 1-2 and 4-5 mum diameter fractions. Isolated microbubbles were stable over two days. After two weeks, however, more dilute suspensions (<1 vol%) were susceptible to Ostwald ripening. For example, 4-5 mum microbubbles disintegrated into 1-2 mum microbubbles. This latter observation indicated the existence of an optimally stable diameter in the 1-2 mum range for these lipid-coated microbubbles. Overall, Differential Centrifugation provided a rapid and robust means for size selection and reduced polydispersity of lipid-coated microbubbles.
Jameel A Feshitan - One of the best experts on this subject based on the ideXlab platform.
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microbubble size isolation by Differential Centrifugation
Journal of Colloid and Interface Science, 2009Co-Authors: Jameel A Feshitan, Cherry C Chen, James J Kwan, Mark A BordenAbstract:Abstract Microbubbles used as contrast agents for ultrasound imaging, vectors for targeted drug delivery and vehicles for metabolic gas transport require better size control for improved performance. Mechanical agitation is the only method currently available to produce microbubbles in sufficient yields for biomedical applications, but the emulsions tend to be polydisperse. Herein, we describe a study to generate lipid-coated, perfluorobutane-filled microbubbles and isolate their size fractions based on migration in a centrifugal field. Polydispersity of the freshly sonicated suspension was characterized by particle sizing and counting through light obscuration/scattering and electrical impedance sensing, fluorescence and bright-field microscopy and flow cytometry. We found that the size distribution was multimodal. Smaller microbubbles were more abundant. Differential Centrifugation was used to successfully isolate the 1–2 and 4–5 μm diameter fractions. Isolated microbubbles were stable over two days. After two weeks, however, more dilute suspensions (
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microbubble size isolation by Differential Centrifugation
Journal of Colloid and Interface Science, 2009Co-Authors: Jameel A Feshitan, Cherry C Chen, James J Kwan, Mark A BordenAbstract:Microbubbles used as contrast agents for ultrasound imaging, vectors for targeted drug delivery and vehicles for metabolic gas transport require better size control for improved performance. Mechanical agitation is the only method currently available to produce microbubbles in sufficient yields for biomedical applications, but the emulsions tend to be polydisperse. Herein, we describe a study to generate lipid-coated, perfluorobutane-filled microbubbles and isolate their size fractions based on migration in a centrifugal field. Polydispersity of the freshly sonicated suspension was characterized by particle sizing and counting through light obscuration/scattering and electrical impedance sensing, fluorescence and bright-field microscopy and flow cytometry. We found that the size distribution was multimodal. Smaller microbubbles were more abundant. Differential Centrifugation was used to successfully isolate the 1-2 and 4-5 mum diameter fractions. Isolated microbubbles were stable over two days. After two weeks, however, more dilute suspensions (<1 vol%) were susceptible to Ostwald ripening. For example, 4-5 mum microbubbles disintegrated into 1-2 mum microbubbles. This latter observation indicated the existence of an optimally stable diameter in the 1-2 mum range for these lipid-coated microbubbles. Overall, Differential Centrifugation provided a rapid and robust means for size selection and reduced polydispersity of lipid-coated microbubbles.
Russell M. Morphew - One of the best experts on this subject based on the ideXlab platform.
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Topography profiles and micrographs of extracellular vesicles purified via Differential Centrifugation and size exclusion chromatography.
2019Co-Authors: Chelsea N. Davis, Helen Phillips, John J. Tomes, Martin T. Swain, Toby J. Wilkinson, Peter M. Brophy, Russell M. MorphewAbstract:Representative atomic force microscopy topography profiles (A and B) and transmission electron microscopy images (C and D) of extracellular vesicles purified from adult F. hepatica via Differential Centrifugation (A and C) and size exclusion chromatography (B and D). The central graphs represent individual extracellular vesicle topography from each purification method as determined by atomic force microscopy. Profiles identified that extracellular vesicle structures were similar using both Differential Centrifugation and size exclusion chromatography purification methods although size exclusion chromatography purified extracellular vesicles were significantly smaller (76 nm ± 44 SD) than Differential Centrifugation extracellular vesicles (95 nm ± 58 SD) (W = 14,726, p < 0.001) and Differential Centrifugation purified extracellular vesicles with a greater range of extracellular vesicle sizes than size exclusion chromatography (Differential Centrifugation range = 505 nm, size exclusion chromatography range = 285 nm). Arrows on TEM images highlight different sized EVs where A = 130.5 nm, B = 235.0 nm, C = 111.7 nm and D = 226.4 nm.
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Protein quantification of extracellular vesicle preparation fractions from Differential Centrifugation and size exclusion chromatography purification.
2019Co-Authors: Chelsea N. Davis, Helen Phillips, John J. Tomes, Martin T. Swain, Toby J. Wilkinson, Peter M. Brophy, Russell M. MorphewAbstract:Fractions produced from either Differential Centrifugation or size exclusion chromatography methods (excretory-secretory protein, whole lysed extracellular vesicle, soluble extracellular vesicle protein and insoluble extracellular vesicle protein) were assayed for protein levels and statistically analysed (Kruskal-Wallis test, with Dunn’s Post-hoc test using Sidak correction) for differences. Asterisks identify significance where p < 0.05.
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Extracellular vesicle protein preparation Western blot profiles purified using Differential Centrifugation and size exclusion chromatography.
2019Co-Authors: Chelsea N. Davis, Helen Phillips, John J. Tomes, Martin T. Swain, Toby J. Wilkinson, Peter M. Brophy, Russell M. MorphewAbstract:Extracellular vesicle preparation western blot profiles of somatic (10 μg protein), excretory-secretory protein (10 μg protein), soluble extracellular vesicle protein (20 μg protein) and insoluble extracellular vesicle protein (20 μg protein). Biological triplicates were analysed on 12.5% SDS-PAGE electrophoresis gels and transferred to nitrocellulose membranes for immune recognition. Samples of extracellular vesicle preparations purified using either the Differential Centrifugation method or size exclusion chromatography method were probed with anti-glutathione transferase sigma class (Anti-FhGST-S1), Anti-Fasciola cathepsin L1 (Anti-FhCat-L1) or anti-fatty acid binding protein V (Anti-FhFABP-V).
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The importance of extracellular vesicle purification for downstream analysis: A comparison of Differential Centrifugation and size exclusion chromatography for helminth pathogens
2019Co-Authors: Chelsea N. Davis, Helen Phillips, John J. Tomes, Martin T. Swain, Toby J. Wilkinson, Peter M. Brophy, Russell M. MorphewAbstract:BackgroundRobust protocols for the isolation of extracellular vesicles (EVs) from the rest of their excretory-secretory products are necessary for downstream studies and application development. The most widely used purification method of EVs for helminth pathogens is currently Differential Centrifugation (DC). In contrast, size exclusion chromatography (SEC) has been included in the purification pipeline for EVs from other pathogens, highlighting there is not an agreed research community ‘gold standard’ for EV isolation. In this case study, Fasciola hepatica from natural populations were cultured in order to collect EVs from culture media and evaluate a SEC or DC approach to pathogen helminth EV purification.Methodology/Principal findingsTransmission electron and atomic force microscopy demonstrated that EVs prepared by SEC were both smaller in size and less diverse than EV resolved by DC. Protein quantification and Western blotting further demonstrated that SEC purification realised a higher EV purity to free excretory-secretory protein (ESP) yield ratio compared to DC approaches as evident by the reduction of soluble free cathepsin L proteases in SEC EV preparations. Proteomic analysis further highlighted DC contamination from ESP as shown by an increased diversity of protein identifications and unique peptide hits in DC EVs as compared to SEC EVs. In addition, SEC purified EVs contained less tegumental based proteins than DC purified EVs.Conclusions/SignificanceThe data suggests that DC and SEC purification methods do not isolate equivalent EV population profiles and caution should be taken in the choice of EV purification utilised, with certain protocols for DC preparations including more free ES proteins and tegumental artefacts. We propose that SEC methods should be used for EV purification prior to downstream studies.
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Protein profiles of extracellular vesicle fractions from Differential Centrifugation and size exclusion chromatography purification.
2019Co-Authors: Chelsea N. Davis, Helen Phillips, John J. Tomes, Martin T. Swain, Toby J. Wilkinson, Peter M. Brophy, Russell M. MorphewAbstract:Biological triplicate protein profiles as observed by one dimensional SDS-PAGE electrophoresis of excretory-secretory protein (10 μg protein), whole lysed extracellular vesicle (10 μg protein), soluble extracellular vesicle protein (20 μg protein) and insoluble extracellular vesicle protein (20 μg protein). All proteins were run on 12.5% SDS-PAGE electrophoresis gels and Coomassie blue stained.
U Ugalde - One of the best experts on this subject based on the ideXlab platform.
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purification of functionally sealed cytoplasmic side out plasma membrane vesicles from saccharomyces cerevisiae
Analytical Biochemistry, 1995Co-Authors: A Menendez, C Larsson, U UgaldeAbstract:Abstract Highly purified plasma membrane vesicles were prepared from yeast protoplasts by a combination of osmotic lysis, Differential Centrifugation, and separation in an aqueous dextran/polyethylene glycol two-phase system. The vesicles were predominantly (85-90%) of cytoplasmic side-out orientation and displayed large ATP-dependent proton pumping activity which was inhibited by vanadate (100 μM) but not by bafilomycin or nitrate. The preparation presented a distinct polypeptide profile with respect to the total membrane fraction and was enriched in the 110-kDa polypeptide corresponding to the plasma membrane H+-ATPase. This preparation of native plasma membranes vesicles is especially suitable for functional studies in vitro.
James J Kwan - One of the best experts on this subject based on the ideXlab platform.
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microbubble size isolation by Differential Centrifugation
Journal of Colloid and Interface Science, 2009Co-Authors: Jameel A Feshitan, Cherry C Chen, James J Kwan, Mark A BordenAbstract:Abstract Microbubbles used as contrast agents for ultrasound imaging, vectors for targeted drug delivery and vehicles for metabolic gas transport require better size control for improved performance. Mechanical agitation is the only method currently available to produce microbubbles in sufficient yields for biomedical applications, but the emulsions tend to be polydisperse. Herein, we describe a study to generate lipid-coated, perfluorobutane-filled microbubbles and isolate their size fractions based on migration in a centrifugal field. Polydispersity of the freshly sonicated suspension was characterized by particle sizing and counting through light obscuration/scattering and electrical impedance sensing, fluorescence and bright-field microscopy and flow cytometry. We found that the size distribution was multimodal. Smaller microbubbles were more abundant. Differential Centrifugation was used to successfully isolate the 1–2 and 4–5 μm diameter fractions. Isolated microbubbles were stable over two days. After two weeks, however, more dilute suspensions (
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microbubble size isolation by Differential Centrifugation
Journal of Colloid and Interface Science, 2009Co-Authors: Jameel A Feshitan, Cherry C Chen, James J Kwan, Mark A BordenAbstract:Microbubbles used as contrast agents for ultrasound imaging, vectors for targeted drug delivery and vehicles for metabolic gas transport require better size control for improved performance. Mechanical agitation is the only method currently available to produce microbubbles in sufficient yields for biomedical applications, but the emulsions tend to be polydisperse. Herein, we describe a study to generate lipid-coated, perfluorobutane-filled microbubbles and isolate their size fractions based on migration in a centrifugal field. Polydispersity of the freshly sonicated suspension was characterized by particle sizing and counting through light obscuration/scattering and electrical impedance sensing, fluorescence and bright-field microscopy and flow cytometry. We found that the size distribution was multimodal. Smaller microbubbles were more abundant. Differential Centrifugation was used to successfully isolate the 1-2 and 4-5 mum diameter fractions. Isolated microbubbles were stable over two days. After two weeks, however, more dilute suspensions (<1 vol%) were susceptible to Ostwald ripening. For example, 4-5 mum microbubbles disintegrated into 1-2 mum microbubbles. This latter observation indicated the existence of an optimally stable diameter in the 1-2 mum range for these lipid-coated microbubbles. Overall, Differential Centrifugation provided a rapid and robust means for size selection and reduced polydispersity of lipid-coated microbubbles.