Presentation Title

Isolation and Quantification of Microvesicles Using Size Exclusion High Performance Liquid Chromatography (SEC-HPLC)

Format of Presentation

Poster to be presented the Friday of the conference

Abstract

Small extracellular vesicles are lipid bilayer enclosed constructs that are released by most cells in a variety of different environments through the process of exocytosis(Xu et al., 2016). The importance of these vesicles can been seen in cellular signalling, cellular environment modification and molecular processes in diseases such as cancer and cardiovascular disorders (Sáenz-Cuesta et al., 2015). Efficient and effective methods for isolating and quantifying high-quality extracellular vesicle samples is crucial for effective use and manipulation of these vesicles. To date there is no cost-effective, easy and standard method for the isolation of these vesicles. Therefore, the purpose of this study is to use HPLC size-exclusion chromatography to replicate a novel isolation protocol (Huang et al., 2015) in an attempt to establish a routine method to isolate and quantify these vesicles. These SEV’s range in size from 40-300nm (Xu et al., 2016); therefore, Sepharose CL-2B will be used as the size-exclusion medium due to the nature of the pore sizes formed in the Sepharose matrix. The vesicles used were isolated from human plasma samples, centrifuged (Sáenz-Cuesta et al., 2015) and applied to the size exclusion column using a high performance liquid chromatography instrument. The presence of cellular contents in the eluate was determined using a UV detector within the HPLC. Further specific identification of the isolated microvesicles will be determined using FITC conjugated anti-human CD41 antibodies and fluorescence detection. These methods will yield a routine and easy method to isolate and quantify extracellular vesicles, which is critical to obtaining high quality samples for further characterization.

Department

Biological Sciences

Faculty Advisor

Mark Rakobowchuk

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Isolation and Quantification of Microvesicles Using Size Exclusion High Performance Liquid Chromatography (SEC-HPLC)

Small extracellular vesicles are lipid bilayer enclosed constructs that are released by most cells in a variety of different environments through the process of exocytosis(Xu et al., 2016). The importance of these vesicles can been seen in cellular signalling, cellular environment modification and molecular processes in diseases such as cancer and cardiovascular disorders (Sáenz-Cuesta et al., 2015). Efficient and effective methods for isolating and quantifying high-quality extracellular vesicle samples is crucial for effective use and manipulation of these vesicles. To date there is no cost-effective, easy and standard method for the isolation of these vesicles. Therefore, the purpose of this study is to use HPLC size-exclusion chromatography to replicate a novel isolation protocol (Huang et al., 2015) in an attempt to establish a routine method to isolate and quantify these vesicles. These SEV’s range in size from 40-300nm (Xu et al., 2016); therefore, Sepharose CL-2B will be used as the size-exclusion medium due to the nature of the pore sizes formed in the Sepharose matrix. The vesicles used were isolated from human plasma samples, centrifuged (Sáenz-Cuesta et al., 2015) and applied to the size exclusion column using a high performance liquid chromatography instrument. The presence of cellular contents in the eluate was determined using a UV detector within the HPLC. Further specific identification of the isolated microvesicles will be determined using FITC conjugated anti-human CD41 antibodies and fluorescence detection. These methods will yield a routine and easy method to isolate and quantify extracellular vesicles, which is critical to obtaining high quality samples for further characterization.