APPLICATION NOTE

CytoFLEX nano Flow Cytometer: the new frontier ofnanoscale Flow Cytometry
Fanuel Messaggio, PhD
Beckman Coulter Life Sciences Headquarters, Indianapolis, Indiana, US.
Email: FMESSAGGIO@beckman.com

CytoFLEX nano Flow Cytometer provides high sensitivity and high resolution, enabling detection andcharacterization of nanoparticles at least as small as 40nm (based on polystyrene beads), accurately
distinguishing particles of similar size within 10 nm difference (based on silica beads), and the ability tocharacterize low-abundance targets in a heterogeneous population.

Introduction
Nanoscale flow cytometry has numerous research applications, and it holds promise in the development
of diagnostic tools and targeted drug delivery systems. The ability to analyze nanoparticles based on
their characteristics opens new possibilities for personalized medicine and nanomedicine.
Although nanoscale flow cytometry is a powerful technique with numerous advantages, it also has
limitations, including detection sensitivity and size resolution. Researchers have been trying to solve thisproblem by stretching available flow cytometers to their lower limit of detection, improving cleaning
techniques and adding cleanup steps in sample preparation, as well as multiple experimental controls
and orthogonal check points.
CytoFLEX nano Flow Cytometer brings nanoscale flow cytometry to a new level, allowing flexibility todesign experiments, thanks to 5 side scatter parameters and 6 fluorescence channels. Performance
consistency is ensured via detailed QC and a fluorescence Sensitivity Monitor tool, as well as multiple
options of on-board cleaning. Here we show an example of 5-color panel on PPP (Platelet Poor Plasma)
EVs isolated from healthy donors.

ProtocolPlatelet Poor Plasma EVs were prepared from whole blood collected in K3-EDTA vacutainers
through a centrifugation at 160 x g for 5 min, followed by a 0.2 μm filtration step and size exclusion
chromatography (SEC) using qEV single 70 nm (Izon). Fractions 4 to 12 were tested on the CytoFLEX
nano analyzer, and one was selected to proceed based on the amount of extracellular vesicles present,
to help ensure a high enough dilution post staining, and to remove antibody aggregates and othercontaminants. Antibodies were spun at 20,000 x g for 10 min at room temperature to eliminate
aggregates, and single antibody titration was run and adjusted for fluorescent spillover. Compensation
was applied to the sample, using the compensation matrix.

Every Event Matters| 1

Figure 1. CytExpert nano software acquisition and compensation matrix settings.

Results
CytoFLEX nano Flow Cytometer enables researchers to effectively detect and resolve plasma-derived
EVs as low as 70 nm (as defined limit by SEC qEV single 70 nm from Izon). This analyzer detects anddistinguish more than one FL signal on single PPP EV particles, including PB, FITC, PE, APC, and PC7,allowing users to see even small positive populations for markers that are low in expression, like CD63
tetraspanin and CD235a for erythrocyte contamination. It allows clear detection without interferenceby antibody background noise. This level of sensitivity can greatly benefit EV research, as most flow
cytometers have been known to be unable to detect EVs smaller than 100nm in diameter.

Figure 2. 5-color panel on PPP EVs isolated from a healthy donor. A. single color control for CD81 PB, CD9 FITC, CD235a PE, CD63 APCand CD61 PC7. B. sample stained with panel showing CD81, CD9 and a low CD63 positivity. CD235a shows a minimal contamination byerythrocytes and CD61 defined platelet derived EVs.

Every Event Matters | 2

While flow cytometry is a tool that provides unique sensitivity and single particle characterization, thedata is expressed in arbitrary units. As the use of flow cytometry evolves in this space, researchers
will require data to be expressed in absolute units such as nanometers and mean equivalent soluble
fluorescence.To accomplish this each researcher will need to select an appropriate method to determine
the size and abundance of fluorescence from each detected particle. Thankfully, robust tools such asFCMpass1 already exist to aid the researcher in creating data that can be compared across time andmultiple orthogonal methods.

REFERENCE
1.

Welsh, J. A. et al. FCMPASS Software Aids Extracellular Vesicle Light Scatter Standardization.
Cytometry A 97, 569–581 (2019).

CytoFLEX nano Flow Cytometer is for research use only. Not for use in diagnostic procedures.
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