APPLICATION NOTE

A new approach to nanoscale flow cytometry with the
CytoFLEX nano analyzer

Background
Nanoscale flow cytometry is a cutting-edge technique that combines the principles of flow cytometry
with nanotechnology. It enables the analysis of particles at the nanoscale, providing valuable
information about their size, composition, and surface properties. Nanoscale flow cytometry has
numerous applications in various fields, including biology, medicine, and materials science. It allows
researchers to analyze and characterize nanoparticles, extracellular vesicles (EVs), and other small
particles with great precision and sensitivity. In addition to its research applications, nanoscale flow
cytometry also 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.
While nanoscale flow cytometry is a powerful technique with numerous advantages, it also has some
limitations, including:Detection sensitivity: there are still limits to the smallest particles that can be detected reliably.
Currently, flow cytometers are not designed to detect and characterize EVs smaller than 100
nm. The detection threshold for nanoparticles is influenced by factors such as background noise,
autofluorescence, and the efficiency of labeling or detection probes.
Size resolution: Flow cytometry can characterize particles in the nanoscale range, but accurately
resolving nanoparticles of similar sizes can be challenging. Distinguishing particles with very small
size differences, such as distinguishing between 50 nm and 60 nm particles, may be difficult due tolimitations in the detection and resolution capabilities of the instrument.
Sample preparation: Preparing samples for nanoparticle or EV detection can be complex. Sample
preparation methods need to consider factors such as aggregation, stability, and potential alterationsof particle properties during the process. Obtaining a representative and homogeneous sample can be
crucial for accurate analysis.
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Standardization: Nanoscale flow cytometry is a relatively new and evolving field, and the field isstill working on standardizing protocols and reference materials. This can lead to variability in datacollection and analysis across different laboratories, making it challenging to compare results andestablish consistent methodologies.
Data analysis complexity: Flow cytometry generates complex and multidimensional datasets, which
require sophisticated data analysis techniques. Analyzing and interpreting large datasets can be timeconsuming, requiring expertise in data processing, visualization, and statistical analysis.
In this application note, we will introduce the CytoFLEX nano Flow Cytometer and its workflow. This
new analyzer is the first purpose-built nanoscale flow cytometer that can detect nanoparticles, such as
extracellular vesicles (EVs) at least as small as 40 nm, while simultaneously performing multiparameter
fluorescent detection. Furthermore, it enables counting, characterization, and particle size definition, allwithin a single instrument, setting a new standard and overcoming current limitations for nanoparticle
research. The CytoFLEX nano software interface, CytExpert nano, provides the sophistication to explore
the unknown at the nanoscale range while providing the ease-of-use characteristics of the CytoFLEX
platform. This way, getting answers to challenging research questions becomes easier than ever for EV
researchers.
The CytoFLEX nano Flow Cytometer offers:
1.

High sensitivity to detect and characterize nanoparticles at least as small as 40 nm (based onpolystyrene beads).

2.

High resolution to accurately distinguish particles of similar size within 10 nm difference (based on
silica beads), and ability to characterize low-abundance targets in a heterogeneous population.

3.

Consistency in instrument performance and data analysis, thanks to a very detailed QC process andfluorescence Sensitivity Monitor, as well as multiple options for on-board cleaning.

4.

Flexibility to design experiments, thanks to 5 side scatter parameters and 6 fluorescence channels.

Introduction
Extracellular vesicles (EVs) are small membrane-bound particles released by cells into the extracellularenvironment. They play important roles in intercellular communication and are involved in various
physiological and pathological processes. EVs are classified into different subtypes based on theirbiogenesis and size, including exosomes, microvesicles, and apoptotic bodies.
Exosomes or small EVs typically range in size from 30 to 150 nm. They are formed through the inwardbudding of multivesicular bodies (MVBs) within the cell, which then fuse with the plasma membrane,
releasing the exosomes into the extracellular space. Exosomes contain various bioactive molecules, suchas proteins, lipids, nucleic acids (DNA, RNA), and microRNAs, which can be transferred to target cells,
influencing their function and behavior.
Microvesicles, also known as large EVs or microparticles or ectosomes, are larger than exosomes,
ranging from 100 to 1000 nm in size. Unlike exosomes, microvesicles are formed by the outward
budding and shedding of the plasma membrane directly. They also carry a diverse cargo of proteins,
lipids, and nucleic acids, and can transfer these molecules to recipient cells.
Apoptotic bodies are the largest EVs, typically ranging from 1 to 5 µm. They are released from dying
cells during the process of programmed cell death (apoptosis). Apoptotic bodies contain cellularfragments, organelles, and nuclear material, and are recognized and engulfed by phagocytic cells tofacilitate their clearance.

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EVs have gained significant attention in recent years due to their potential as biomarkers for diseasediagnosis and prognosis, as well as their roles in cell-to-cell communication and their therapeutic
applications. Researchers are studying EVs in various biological fluids, including blood, urine, andcerebrospinal fluid, to gain insights into their cargo and functions.
Technologies such as nano flow cytometry, electron microscopy, and molecular profiling techniques likeRNA sequencing and proteomics are used to study and characterize EVs.
Understanding EV biology, cargo, and functions holds great promise for advancing our knowledgeof cellular communication and their potential applications in diagnostics, therapeutics, andregenerative medicine.
Currently, EV analysis is critical and challenging. Isolation and purification methods can suffer fromlow yield, contamination from other particles, and difficulties in standardization. Heterogeneity of EV
populations in terms of size, cargo, and biogenesis complicates their study. EVs can exhibit diverse
biological activities and functions depending on their cellular origin and cargo. However, deciphering
the specific functions and mechanisms of action of EVs in different contexts is still a challenge. The
functional heterogeneity of EVs requires more comprehensive characterization and standardized
functional assays.
Researchers in the field of EVs are actively working to address these limitations by developing improved
isolation techniques, standardizing protocols, and advancing our understanding of EV biology. As thefield progresses, overcoming these challenges will help unlock the full potential of EV research and its
applications in various biomedical areas.
One of the biggest limitations is the need for multiple techniques to accurately count, characterize anddetermine the size of EVs, resulting in time-consuming and laborious workflows with poor repeatability.
The CytoFLEX nano Flow Cytometer combines everything in only one instrument, enabling count,characterization, and size determination, thanks to the following features:
Performance
Violet Side scatter sensitivity
Scatter Detection dynamic range
Violet forward scatter sensitivity

VSSC1: 40 nm relative to polystyrene nanoparticles
VSSC2: 80 nm relative to polystyrene nanoparticles
VSSC1 for small range 40-150 nm polystyrene nanoparticles
VSSC2 for large range 80-1000 nm polystyrene nanoparticles
300 nm relative to polystyrene nanoparticles
Simultaneous detection of fluorescence on six fluorescence detectors,
using 500 nm CytoFLEX nano Multi-intensity Fluorospheres
V447: 8 peaks
B531: 8 peaks
Y595: 8 peaks
R670: 6 peaks
R710: 5 peaks
R792: 4 peaks

Fluorescence sensitivity and resolution

Fluorescence rCV

rCV <10% (using QC Fluorospheres at 1 µL/min)≤ 1% aCarryover between samples
Acquisition speed

Maximum electronic acquisition speed

16000 events/second with ≥95% yield

Recommended maximum sample
acquisition speed

5000 events/second to avoid possible
swarming or coincidence situation

Volumetric counting accuracy≥90% btested with polystyrene beads, b tested with 144nm QC Scatterspheres on 3 units under the conditions: record 3 minutes at the sample
flow rates of 1 µL/min, 2 µL/min, 3 µL/min, 4 µL/min, 5 µL/min, 6 µL/min respectively, repeat 5 times, then calculated the average total
events for each speed, compared the calculated the volumetric counting accuracy with the theoretical total events.
aFigure 1. The CytoFLEX nano Flow Cytometer performance specifications.

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Protocol
1.

System Startup

After comfirming that the sheath fluid and the cleaning reagent are sufficient for the day, and waste
container is empty, turn on the CytoFLEX nano Flow Cytometer and start the CytExpert nano software,using the link on the desktop. Select the System Startup procedure. The procedure will take about 6
minutes, during which the system automatically purges the sheath damper, executes debubble for the
sheath filter, the sheath line, the flow cell and, the piston pump, and cleans the sample line, ensuring thefluidics system is set to start.
2.

Configuration setting

The CytoFLEX nano Flow Cytometer is equipped with 2 Wavelength Division Multiplexers (WDMs),
one for scatter optical filters (VioletSSC1, VioletSSC2, BlueSSC, YellowSSC and RedSSC) and one forfluorescence optical filters (V447, B531, Y595, R670, R710, R792), as shown in Figure 2 below.

Figure 2. The CytoFLEX nano Flow Cytometer default detector configuration.

3.

Perform Daily QC

Select the QC/Sensitivity menu and input the bead lots. Running QC will ensure the CytoFLEX nano
Flow Cytometer will provide adequate signal strength and precision.
The QC process will start evaluating laser powers and sheath flow rate.
Next, the process is divided in three steps:• Instrument background assessment: using 5 nm filtered CytoFLEX sheath the system assesses
background noise in both sheath and sample line, very close to the limit of optical and electricalnoise, to ensure it will achieve the lowest limit of detection.
• Scatter performance assessment: using CytoFLEX nano Daily QC Scatterspheres the system will
monitor event rate and signal strength.
• Fluorescence performance assessment: using CytoFLEX nano Daily QC Fluorospheres thesystem will monitor event rate, laser delay and fluorescence detection performance. Between
each tube the system will trigger an automatic backflush cycle with sheath fluid. The generated
QC report confirms success at each step. If a failure occurs, it clearly indicates what needs
attention.

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4.

Sensitivity Monitor

When fluorescence sensitivity is key, the sensitivity monitor will add confidence, showing the numberof fluorescence peaks the CytoFLEX nano Flow Cytometer can resolve and the distance between thenoise and dimmest resolvable peak. Select Sensitivity monitor in the QC/Sensitivity menu and input the
lot you will be using. This process will run the Multi-fluorescent Fluorospheres, which are a mixture ofmulti-intensity multi-fluorescent 500 nm polystyrene beads. See Figure 3 for an example of the QC and
Sensitivity Monitor Reports.

Figure 3. Example of QC and Sensitivity Monitor Reports.

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5.

The CytoFLEX nano Flow Cytometer on-board clean processes

The CytoFLEX nano Flow Cytometer offers multiple automated cleaning options to meet different
sample and workflow needs. Each selected workflow ensures that researchers can accurately andreproducibly characterize EVs and other biological nanoparticles, confirming that the background noise
does not impact EV/nanoparticle evaluations.
Workflows available:• Backflush is automatically integrated into Unload process, and it will run after QC and Sensitivity
Monitor. It can also be triggered by clicking on Backflush in the Acquisition Control Panel. In
the Cytometer menu, select Cytometer Configuration and select the number of backflushes the
instrument will run when Backflush will be initiated.
• On-board Clean has 3 pre-set options in the Cytometer Configuration menu and a customized
number of back and forth (BFF) with CytoFLEX cleaner at the end:
— Option 1: 1 BFF (cycle time: 7min 19s)
— Option 2: 5 BFF (cycle time: 10min 26s)
— Option 3: 10 BFF (cycle time: 14min 18s)
— Option 4: Custom - Select Back & Forth Cycles, BFF from 1 to 10. Corresponding time willappear.
• Manual Clean is a cleaning process similar to what the CytoFLEX instruments Daily Clean offers.
When the time of Manual Clean is set similar to On-Board Clean, the results are comparable.
Selecting Manual Clean from the Cytometer menu, the following steps are to define the time withcleaner and time with water or sample buffer.
• Shutdown Clean— Option 1: is to perform the selected On-board Clean and shutdown automaticallyafterwards.
— Overnight (recommended 8 hours) soaking of the sample line is expected to completely
clear the line. To extend flexibility, soaking can be stopped at any time.
• Flow Cell Clean is necessary only in extreme cases, which a service representative can help
define. The process run with freshly prepared 10% Contrad 70 solution, placed in its onboard
bottle, in the front of the CytoFLEX nano Flow Cytometer.
— Select Flow Cell Clean on the Cytometer Menu. The system rinses and soaks the Flow Cellwith 10% Contrad 70 solution. The soaking is suggested to be at least 30min. Following thatthe instrument proceeds to rinse out Contrad 70 with multiple Flow Cell Primes.
— Flow Cell Prime (in the Cytometer menu) can be selected and used outside of the Flow Cell
Clean, and it is useful when detergents are used.

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Figure 4. Backflush and On-Board Cleaning Setting.

6.

Experiment Setting• Select File and open a new experiment.
• On Cytometer Menu, select Cytometer configuration and input the volume of sample that will becollected by the sample pump from the sample tube. There is 32 µL of dead volume to consider,and the minimum volume in the sample tube is 100 µL.
• On Cytometer Menu, select Settings and Options. Sample volume is enabled by default, so thesystem will monitor sample consumption and stop automatically when there is less than thedefine volume to collect. The sample volume monitor can be disabled by unchecking that box.
• On the Acquisition Control Panel on the left, select:— How many events to display and the stopping rule for recording. It can be by a numberof events in a specific gate, by time or by volume. If multiple options are selected the
instrument will stop whichever will come first.
— Sample flow rate. 1 µL/min is suggested for beads acquisition and 3 µL/min is suggested forbiological samples. Other options available are 6 µL/min and customized between 1 and6 µL/min.
• Use the icons on icon top bar to generate the type of histograms and plots needed for theexperiment. On that menu, there are also statistics, hierarchical gating, manual and automatedgates, scaling, gain and compensation tools.

7.

Sizing calibration

To obtain reliable and accurate measurements, sizing calibration is needed. NanoVis product is a multisize mix of polystyrene beads, including 44 nm, 80 nm, 100 nm 144 nm, 300 nm, 600 nm, and 1 µm.
Thresholding in VSSC1, the mix results assessed by a reference calibration method will enable you togenerate calibrated data with the CytoFLEX nano Flow Cytometer.

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8.

Sample Buffer and Baseline Monitor Setting• Select a new tube, using theicon in the Acquisition Control Panel on the left, and load a tube
of sample buffer. Click run and proceed to Acquisition Setting. From this menu, adjust threshold
and gain of all the interested channels for the ongoing experiment. This step will create a baseline
for the future samples that will be run.
• The sample buffer tube can be used to generate baseline monitor Settings. Select a new Blank,
using theicon in the Acquisition Control Panel on the left. Right click on the tube and selectBaseline Monitor. In the menu select:— Blank sample type: sample tube will require a sample buffer tube in the sample holder
every time baseline monitor will be run, where sample line will trigger a pull in of 5 nm
sheath in the sample line.
— Acceptance criteria: events/sec or a number of events in all events or a specific gate drawn.
The gate can be drawn at the right of the noise in the most sensitive scatter channel, VSSC1.
This specific gate will monitor any residue in the sample line, other than the noise comingfrom the sample buffer, allowing a measure of the instrument readiness for the following
sample.
— Cleaning Cycle Settings: if the acceptance criteria are not met, define how many cycles
of backflush and how many backflushes per cycle will be run. Checking On-Board Clean
box will include a cycle of selected On-Board Clean. Between each cleaning cycle theinstrument will reassess baseline and if the acceptance criteria are met. If yes, the monitor
will stop; if not the following defined cleaning cycle will be run.
— Baseline monitor will be saved as fcs file: All Cycles Data will merge all the baseline monitor
run in one fcs file, where Last Cycle Data will save only the last run.
— Once set, Baseline Monitor, with these settings, can be initiated during the experiment bygenerating a new Blank tube and clicking Run. Setting can be modified by right clicking on
the blank tube and selecting Baseline Monitor.

Figure 5. Baseline Monitor Setting.

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9.

Sample acquisition• Using theicon in the Acquisition Control Panel on the left, create a tube with the same settingof the sample buffer, and load a sample tube.
• When working with a new sample type, start with a sample titration, running from the lowest
to the highest concentration. A slight modification of gain and threshold can be applied to best
resolve the sample. If that is the case, we suggest repeating the sample buffer run and changing
the Baseline Monitor Setting.
• Coincidence and swarming, as cellular flow cytometry, depend on sample concentration but for
the CytoFLEX nano Flow Cytometer, it is also dependent on particle size. For samples with a size
higher than 100-150 nm, the order of 104 particles/µL can be considered a good concentration
and a limit before swarming. The more representation of small particles (and/or higher the small
EV/large EV ratio) allows a higher concentration to be run. If swarming conditions are too highthe Events Processed % in the Acquisition Control Panel will be lower than 100%.
• Once the best sample dilution is selected, based on the number of events per second or in a
specific gate, and sample resolution and representation in the plots of interest, start running
single-color stained samples and adjust fluorescence gains in the acquisition setting menu.
• When a panel of color will be run, compensation can help correct for fluorescence spillover,removing undesired signal. Compensation can be done using the Compensation Matrix, selecting
theicon in the Acquisition control panel, or done manually using theicon in the top bar.
• CytoFLEX nano Flow Cytometer offers fluorescent triggering options that can help when
focusing on specific stained/labeled populations.
• Baseline Monitor is suggested between different sample types, or when a lower concentration
will be run, or when an unstained sample will be run after a stained one.
• Always run all controls as suggested by MIFlowCyt-EV position paper (ref #1). If a detergenttreated sample will be run, we suggest a detergent titration first, starting from the lowest
concentration. This will evaluate the detergent background on CytoFLEX nano Flow Cytometer.
When multiple tubes of detergent or a high concentration will be run, perform debubble options
and Prime to eliminate possible nano- and micro-bubble formation.

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Figure 6. Acquisition setting and Compensation Matrix.

10. Cleaning and System Shutdown Program• Between different experiments, an On-Board Clean is suggested. Select Cytometer in the menubar, followed by On-Board Clean.
• At the end of the day, select Cytometer in the menu bar, followed by System Shutdown program.
The program provides three options for different needs:— Clean the sample line with cleaner with the selected On-Board clean cycle, prior to the
System Shutdown.
— Running long-term soaking prior to the System Shutdown.
— System Shutdown only.

Figure 7. System Shutdown.

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Conclusions
The CytoFLEX nano Flow Cytometer is the first flow cytometer that can clearly demonstrate detectionof extracellular vesicles down to a size of 40 nm (measured with polystyrene beads). Its high sensitivity
and resolution for small particles, combined with automated cleaning, extensive QC process andFluorescence Sensitivity monitor, will undoubtedly propel the field of research forward. The capabilities
and features of this instrument will enable researchers to explore previously uncharted territories andobtain more comprehensive and accurate data. With this cutting-edge tool, we anticipate significant
advancements in research findings and a deeper understanding of extracellular vesicles, and theirapplications.

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Nieuwland R, Wauben MHM, Nolan JP, Jones JC. MIFlowCyt-EV: a framework for standardized
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Cook S, Tang VA, Lannigan J, Jones JC, Welsh JA. Quantitative flow cytometry enables end-toend optimization of cross-platform extracellular vesicle studies. Cell Rep Methods. 2023 Dec
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