Amnis® ImageStream®X Mk II High Gain Mode for Increased Small Particle Detection and EV Analysis on the Amnis® CellStream® Using the vFC™ Assay from Cellarcus Biosciences, Inc. - Luminex Corporation
Amnis® ImageStream®X Mk II High Gain Mode for Increased Small Particle Detection and EV Analysis on the Amnis® CellStream® Using the vFC™ Assay from Cellarcus Biosciences, Inc.
Haley Pugsley, PhD, Manager, Senior Scientist at Luminex Corporation
John Nolan, PhD, CEO, Cellarcus Biosciences, Inc.
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. In this tutorial, we first describe the use of High Gain mode on the Amnis® ImageStream®X Mk II Imaging Flow Cytometer to address the challenges of measuring small particles. In this new High Gain mode, the charge-coupled device (CCD)-camera is 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. The second part of this tutorial describes the use of a quantitative and specific Vesicle Flow Cytometry assay (vFCTM, Cellarcus Biosciences, Inc.) that takes advantage of the high sensitivity of the Amnis® CellStream® Flow Cytometer. The vFCTM assay consists of a full suite of reagents needed to analyze EVs, including stains, calibrators, and standards, as well as detailed sample preparation and analysis protocols. This assay ensures quantitative and reproducible results. Additionally, we describe the measurement of EV numbers, size, and surface cargo, including tetraspanin and cell-specific markers using the Amnis® CellStream®.
CMLE Credit: 1.0