Demonstration of High Gain mode in combination with Imaging Flow Cytometry for improved EV analysis

Abstract

Abstract Extracellular vesicles (EVs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. In recent years, the importance of EVs has become apparent, as they are key mediators of intercellular communication. However, quantifying and characterizing EVs in a reproducible and reliable manner is challenging due to their small size—exosomes range from 30 to 100 nm in diameter. It is well-known that flow cytometers were originally designed to measure and detect cells, and due to the quantitative power flow cytometry offers, there has been a push to quantify and characterize EVs using flow cytometric methods. However, these systems have not been designed to measure objects smaller than a cell. Here, we describe the use of High Gain mode on the Amnis® ImageStream® Imaging Flow Cytometer to address the challenges of measuring small particles. In this new High Gain mode, the charge-coupled device (CCD)-camera is manually adjusted to higher gain settings, increasing the signal obtained from the EV. Object thresholds and masking have also been adjusted to better identify and detect small particles. Preliminary results using murine leukemia virus-sfGFP reference particles have shown up to a 5-fold increase in the number of GFP-positive objects collected in High Gain mode, when compared to standard gain on the ImageStream® System. In this study, we demonstrate improved small particle detection, including EVs, using this new High Gain mode.