Skip to main content

The EV Core facility is engaged in a wide range of research activities related to extracellular vesicles (EVs) with a primary focus on developing and applying advanced technologies and methodologies. A key aspect of our research involves the phenotypic characterization of EVs using fluorescence-labeled antibodies, which are crucial for our studies. Additionally, membrane staining with lipophilic dyes is widely employed. In our protocols, we isolate EVs from various primary biofluids, including blood plasma, urine, and ascites. The handling of these diverse biofluids necessitates customized protocols to ensure the integrity and purity of the isolated EVs, and it requires stringent controls especially when working with the aforementioned dyes.

Here, we demonstrate the efficacy of using fluorescence-labeled CD63 antibodies for the specific tagging of EVs. By employing Vesi-SEC micro columns, we can efficiently remove excess antibodies as well as EVs stained with a lipophilic dye, significantly improving the clarity and reliability of the subsequent nano flow cytometry analysis (Figure 1 & 2). The utilization of Vesi-SEC micro columns significantly enhances the sensitivity and specificity of EV analysis, while requiring minimal hands-on time, enabling more precise and reliable phenotypic characterization.

Figure 1. Enhancement of signal detection in antibody-labeled extracellular vesicles by Vesi-Sec micro column processing. EVs were isolated from ascites fluid using a combination of differential centrifugation and tangential flow filtration, followed by size exclusion chromatography with homemade Sepharose CL-2B 10mL columns. Approximately 5 × 108 particles were subjected to incubation with APC-labeled CD63 antibody (clone H5C6, Biolegend) overnight at 4 °C with constant agitation. The EVs were analyzed by nano-flow cytometry both before (pre) and after (post) processing with the Vesi-Sec micro columns. Bivariate dot plots illustrate the fluorescence intensity (APC) versus side scatter (SSC), highlighting the signal amplification post-processing.

Figure 2. Enhancement of signal detection in lipophilic-dye-labeled extracellular vesicles by Vesi-Sec micro column processing. EVs were isolated from ascites fluid using a combination of differential centrifugation and tangential flow filtration, followed by size exclusion chromatography with homemade Sepharose CL-2B 10mL columns.Approximately 5 × 108 particles were subjected to incubation with a fluorophore-conjugated lipophilic dye clone (CellMask™ Deep Red, Thermo Fisher Scientific) for 1 hour at room temperature. The EVs were analyzed by nano-flow cytometry both before (pre) and after (post) processing with the Vesi-Sec micro columns. Bivariate dot plots illustrate the fluorescence intensity (APC) versus side scatter (SSC), highlighting the signal amplification post-processing.

Dr. Christian Preußer

Philipps University Marburg Center for Tumor Biology and Immunology

EV - Core Facility

For more information on Vesi-SEC micro, please visit our product page.

en_GBEnglish