TABLE 1.

Main differences between flow cytometry (FCM) experiments on cells and extracellular vesicles (EVs). APC: allophycocyanin; CD: cluster of differentiation; FSC: forward scattered light; PE: phycoerythrin; SSC: side scattered light

Chapter	Topic	Cells	EVs
3. Experimental design & pre‐analytical procedures		Cells have a large surface area, express many different proteins and have a large cytosolic volume for intracellular staining compared to EVs. Therefore, it is feasible to detect more than 40 markers simultaneously.	Multiple factors limit the detection of multiple markers on EVs. These include limited surface area resulting in steric hindrance and flow cytometers typically having only 2–3 detectors capable of detecting <10 copies of a fluorophore.
	Fluorophore selection	In most cases the protein expression is high enough that modern flow cytometers can detect most fluorophores. Limitations in fluorophore detection mainly arise when larger fluorescent antibody panels are being built where there is significant spectral overlap and marker abundance needs to be accounted for.	EV detection using FCM requires the use of the brightest possible fluorophores, for example, PE, APC
4. Sample preparation	Fluorescent staining	When stained, cells are bright due to expressing high numbers of receptors, making it easy to stain a known number of receptors and remove unbound dye.	Due to limited numbers of receptors, stained EVs are dim, which hampers the detection of all EVs in a sample as well as antibody titration. In addition, the removal of unbound reagents typically results in dilution and loss of EVs and therefore is difficult, particularly for high‐throughput scenarios.
	Sample washing	Minor cell loss, because cells pellet down efficiently.	EVs may be discarded, because EVs are small and do not pellet down efficiently.
5. Assay controls	Buffer only controls	Cells exceed background noise.	EVs are close to and below the background noise.
	Buffer with reagents controls	Antibody aggregates have signals lower than cells.	Antibody aggregates may have signals similar to EVs.
	Unstained controls	Reagents do not affect the count rate of cells	Reagents may affect the count rate of EVs
	Isotype controls	Non‐specific binding to Fc receptors	Non‐specific binding to Fc receptors
	Single‐stained controls	Spectral spill over	Spectral spill over
	Procedural controls	Staining methods require specific processing steps after staining	Staining methods require specific processing steps after staining
	Serial dilution controls	Flow cytometer electronics are typically designed to detect and remove coincidence events. While two or three events may be coincidentally detected as doublets or triplets, this can usually be removed from analyses using parameter height, width, area comparisons.	Multiple (hundreds or more) EVs may be artefactually counted as one event (swarm detection)
	Detergent treatment controls	N/A	Unclear whether detected particles are envisioned EVs or other particles
6. Instrument data acquisition and calibration	FSC vs. SSC	Cells scatter majority of light in FSC direction. FSC is often used to determine relative size while SSC is a means of determining relative granularity of cells.	EV light scatter becomes more isotropic as they become smaller. FSC is less sensitive to resolve EVs than SSC on the majority of flow cytometers and SSC can be used in combination with Mie modelling to approximate the EV diameter.
	Trigger channel(s) and thresholds	Cells scatter the majority of light in FSC direction and exceed the limit of detection. The FSC threshold is sufficient for majority of cell applications	EVs are not fully detectable by flow cytometers. The best trigger channel and threshold for the maximum signal to noise ratio will depend upon the assay and will likely be an SSC or fluorescent trigger.
	Flow rate/volumetric quantification	Flow rate is generally not a concern for typically cellular analysis given that the input material is typically a diluted to a set concentration for staining for example,106 cells/ml	Flow rate should be carefully considered for EV analysis and the impact of changing the flow rate to the sensitivity should be assessed. In many cases increasing the flow rate can decrease the sensitivity and increase the signal variation.
	Fluorescence calibration	Fluorescence calibration was developed to help determine lymphocyte epitope abundance, for example, CD4 epitopes	Fluorescence calibration is required to determine instrument sensitivity and enable comparisons across platforms with difference limits of detection. Fluorescence calibration is also used for EV characterization, such as epitope abundance.
	Light scatter calibration	Light scatter calibration not required due to the ease of cellular detection on commercial cytometers.	Light scatter calibration is required to determine instrument sensitivity and enable comparisons across platforms with difference limits of detection. Light scatter calibration is also used for EV characterization, such as diameter or refractive index approximation.