Small Particle Detection And Analysis On The ZE5 Cell Analyzer

Exosomes are a type of extracellular vesicle (EV) that contain proteins, RNA, and DNA, and have been implicated in general intracellular communications in health and disease. Exosomes are involved in many processes related to cancer, including tumorigenesis, metastasis, and drug resistance to note just a few (Guo et al. 2017). As messengers, exosomes may contain useful biomarkers, and as such have become a key area of interest in disease research. Due to the small size of exosomes (generally <250 nm), there are limited methods available with which to study them.

The most common method of exosome analysis by flow cytometry is using magnetic beads for capture and detection. Most flow cytometers can be used in this type of analysis because the beads lend detectable size to the samples. The ZE5 Cell Analyzer also has small particle detection capability (forward scatter [FSC] from 405 nm laser), so we wanted to investigate whether we could detect exosomes directly as well as captured on beads. 

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We studied the presence of common exosomal surface markers CD63 and CD81 (Heijnen et al. 1999), and intravesicular markers ALIX and TSG101 (Lee et al. 2012) in exosomes purified from MCF-7, a breast cancer cell line. We also demonstrated the use of  SureBeads to capture exosomes and stain for surface markers. We present both methods of staining and instrument setup, and details for direct exosome detection on a ZE5 using single and dual triggers.