Nanosight software

The concentration and size distribution of 30× concentrated (not purified) VLPs were measured with NanoSight (NS300) (Malvern Panalytical Ltd., Malvern, UK) equipped with NTA software (version 3.4; Malvern Panalytical Ltd., Malvern, UK). All samples were diluted in PBS to a final volume of 1 mL. Ideal measurement concentrations were found by pre-testing the ideal particle per frame value (20–100 particles/frame). For each sample, 5 videos of 60 s were recorded with a Camer Level of 13. After capture, the videos were analyzed by the NanoSight Software (version 3.4; Malvern Panalytical Ltd., Malvern, UK) with a detection threshold of 5. The settings were established according to the manufacturer’s software manual (NanoSight NS300 User Manual, MAN0541-01-EN-00, 2017).

The size distribution and concentration of EVs purified from plasma by UCF, scUCF, Vn96, ME buffer and Scr peptide (from Fig. 1a, Step 6ii) were measured using a NanoSight NS300 equipped with a 405 nm laser (Malvern Panalytical Ltd.,United Kingdom) and analyzed using Nanosight software (v3.2, https://www.malvernpanalytical.com/en/support/product-support/software/NanoSight-NTA-software-update-v3-2, accessed on 28/01/2021). Samples were prepared for NTA analysis by diluting the stock material in particle-free water until the concentration was between 1 × 10⁸ and 1 × 10⁹ particles/mL and six videos of 30 s were captured, analyzed with Gain = 512 and shutter at 1300, and averaged. At least two different dilutions resulting in good correlation of the stock concentration (% coefficient of variance (%CV) < 25%) were included in the analysis for each sample. Size distribution data was analyzed by normalizing the concentration of particles of different diameters with bin widths of 1 nm to the highest concentration observed within a 1 nm bin and then taking the average of each measurement. The average of each technical replicate (n = 2) was averaged with each biological replicate (n = 3) and expressed as particles per mL of plasma ± standard error of the mean (SEM).

MkEVs were diluted by between 25‐ and 35‐fold in filtered PBS and characterized using a NanoSight NS300 instrument (Malvern Panalytical, Malvern, UK) as described previously.¹⁸ Data were analyzed with NanoSight software (Malvern Panalytical, Malvern, UK) using a camera level of 10, a detection threshold of 15, and a syringe pump speed of 60. For each biological replicate, measurements were taken in triplicate and averaged.

EV suspensions were diluted 1:20 or 1:200 to permit counting in the range of 3–15×10⁸ per ml with an NS500 nanoparticle tracking analysis system (NanoSight, Amesbury, UK). The EVs were visualized by their scattering of a focused laser beam and the collection of the scattered light using a standard optical microscope fitted with a CCD video camera. Five exposures of 20 s each were recorded from fields chosen randomly by a computer operating with NanoSight software, Malvern Instruments (Malvern, United Kingdom), which enables measurement of EV size and numbers.

The concentration and size distribution of FBS-EVs were analyzed using a NanoSight NS300 (Malvern Panalytical, Malvern, UK). The laser was set at 532 nm, and three 60-s videos were recorded for each sample at 25 frames/s using an sCMOS camera (Thorlabs, Netwon, NJ, USA). For all video recordings, the camera level was set to 14, with a well-adjusted camera focus, and the detection threshold was set to 5. The Brownian movement of EVs was assessed using the NanoSight software (v3.4; Malvern Panalytical, Malvern, UK). For optimal measurements, NTA settings were maintained consistently between samples diluted with 1× phosphate-buffered saline (PBS).