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Use of flow cytometry to analyze small particles has been implemented for several decades. More recently, small particle analysis has become increasingly utilized owing to the increased sensitivity of conventional and commercially available flow cytometers along with growing interest in small particles such as extracellular vesicles (EVs). Despite an increase in small particle flow cytometry utilization, a lack of standardization in data reporting has resulted in a growing body of literature regarding EVs that cannot be easily interpreted, validated, or reproduced. Methods for fluorescence and light scatter standardization are well established, and the reagents to perform these analyses are commercially available. Here, we describe FCM_PASS, a software package for performing fluorescence and light scatter calibration of small particles while generating standard reports conforming to the MIFlowCyt‐EV standard reporting framework. This article covers the workflow of implementing calibration using FCM_PASSas follows: acquisition of fluorescence and light scatter calibration materials, cataloguing the reference materials for use in the software, creating cytometer databases and datasets to associate calibration data and fcs files, importing fcs files for calibration, inputting fluorescence calibration parameters, inputting light scatter calibration parameters, and applying the calibration to fcs files. Published 2020. U.S. Government.

Basic Protocol 1: Acquisition and gating of light scatter calibration materials

Basic Protocol 2: Acquisition and gating of fluorescence calibration materials

Alternate Protocol: Cross‐calibration of fluorescence reference materials

Basic Protocol 3: Cataloguing light scatter calibration materials

Basic Protocol 4: Cataloguing fluorescence calibration materials

Basic Protocol 5: Creating cytometer databases and datasets

Basic Protocol 6: Importing fcs files

Basic Protocol 7: Fluorescence calibration

Basic Protocol 8: Light scatter calibration

Basic Protocol 9: Performing and reporting fcs file calibration

Extracellular vesicles (EVs) are sub‐micron‐sized membranous spheres secreted by cells. EVs play a functional role as intercellular communicators and are associated with a number of diseases. Research into EVs is an area of growing interest due their many potential uses as therapeutic agents, as diagnostic and theranostic biomarkers, and as regulators of cellular biology. Flow cytometry is a popular method for enumerating and phenotyping EVs, even though the majority of EVs are below the detection sensitivity of most commercially available flow cytometers. Here, we present optimized protocols for EV labeling that increase the signal‐to‐noise ratio of EVs by removing residual antibody. Protocols for alignment of high‐resolution jet‐in‐air flow cytometers are also provided. Published 2020. U.S. Government.

Basic Protocol 1: Bulk EV staining with CFSE protein binding dye

Basic Protocol 2: Antigen‐specific staining of EV markers with fluorochrome‐conjugated antibodies

Basic Protocol 3: Astrios EQ instrument setup and sample acquisition

Basic Protocol 4: Counting particles and EVs on Astrios EQ with spike‐in reference beads