Evaluation of plasmatic extracellular vesicles by size

Extracellular vesicles (EVs) play a key role in many physiological and pathological processes [1]. EVs are a heterogeneous group of membrane-conned particles including endosome-derived exosomes and plasma membrane-originated microvesicles. The expanding eld of extracellular vesicle research needs reproducible and accurate methods to characterize EVs [2]. EV proling can be challenging due to the small size and heterogeneity. This protocol aims to provide a method to isolate EVs and facilitate high-precision particle quantitation by Nanoparticle Tracking Analysis (NTA)[3, 4]. NTA is commonly used to determine EV concentration and diameter [5, 6]. The protocol here described refers to the isolation of EVs from blood-plasma samples by using ultracentrifugation and then quantication and sizing of EVs with NTA by NanoSight NS300 system (Malvern Panalytical Ltd., Malvern, UK) provided with a syringe pump module enabling analysis in constant ow for improved sample statistics.


Introduction
In the last decade, the regulatory role of extracellular vesicles (EVs) in pathological and physiological processes has gained increasing interest [1]. Although several methods Have been developed for the isolation and puri cation of EVs, ultracentrifugation (UC) is still considered the gold standard to isolate EVs. UC is widely used to isolate EVs, from both biological uids (e.g. plasma) and cellular conditioned media [7].
Nanoparticle Tracking Analysis (NTA) is a biophysical method based on optical density tracking of particles. NTA can simultaneously measure the size distribution and concentration of particles, such as EVs, suspended in a liquid. NTA uses laser light scattering microscopy with a Charge-Coupled Device (CCD) or complementary metal-oxide-semiconductor (CMOS) camera. Brie y, based on the Brownian motion of the particles, which are considered as having a spherical shape, the diffusion coe cient of each tracked particle is calculated using the Stokes-Einstein equation; the movement of every single particle is tracked and then used to estimate particle size [8].
This protocol aims to provide a method to isolate EVs from plasma samples and facilitate high-precision particle quantitation by Nanoparticle Tracking Analysis (NTA). Moreover, as the large majority of the papers describing NTA data focus on the mean size or the mean concentration of EVs, we propose a new statistical approach to consider all the sample sizes and a graph designed to report these results.  7. Discard the supernatant using a 10 ml syringe (needle dimension 0.9 mm x 25 mm).

Reagents
8. Dry the wall of the tube using a sterile cotton swab.
3. Use 1mL insulin syringe to carry the sample on the syringe pump. 6. Check the analysis setting before going on with the batch process. The number of particles per frame must be 20-120. Set detection threshold to have < 5 not valid track per frame. The ratio between total particles track and valid track must be < 5.
7. Collected data are analyzed with NTA software (Malvern Panalytical Ltd.), which provided highresolution particle-size distribution pro les as well as measurements of the EVs concentration.

Statistical method to analyze data from Nanosight
To compare EVs concentration for each size in different groups (i.e. cases vs controls) we apply generalized multivariable linear models, using the appropriate logit link function. We estimate adjusted EV mean concentration or EV geometric mean concentration as appropriate. For each size, from 30nm to 700nm, we compare the EV mean differences between groups. Due to the high number of comparisons, we apply a multiple comparison method based on Benjamini-Hochberg False Discovery Rate (FDR) to calculate the FDR P-value. Results are reported as a series graph for EV mean concentrations of each size by group and vertical bar charts to represent the size-speci c p-values obtained comparing groups. For all the graphs, X-axis is the size of EVs. In Figure 2 we report an example of this analysis. Troubleshooting 1. We resuspend the sample with PBS three-time ltered through a 0.10-μm pore-size polyether sulfone lter (Millipore membrane lters) to avoid interference in NTA analysis as background noise.
2. With NTA-acquisition settings constant between analyses, our approach will enable the mean, mode, and median particle size together with EV concentration to be more precisely compared between different samples.
3. Remove the command "Advanced sample prompt" on NTA software, to allow the syringe pump ow to be continuous among the ve recordings.
4. Between samples, push 2 ml of water through the sample chamber, to remove any particles present. After use, the chamber can be disassembled to be cleaned and then reassembled. Rinse the seal and the glass on the underside of the gasket component with a low-pressure source of water and then an up to 10%ethanol-water solution; nally dry with tissue papers without rubbing but gently tapping.

Time Taken
Anticipated Results Figure 2 Panel A: Mean concentrations of EVs (*106) for each size (nm) by case or control subjects. Panel B: Pvalue and False Discovery Rate P-value obtained from the appropriate statistical model (in this example, a Poisson regression models allowing for over dispersion), for each EV size.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. PlasmaEVsNanosight.avi