Nta software version 3

NTA of exosomes was performed using Quantum Design Japan (Tokyo, Japan). Exosome concentration and size distribution were analyzed using NanoSight (LM10V-HS; Malvern Panalytical Ltd., Malvern, UK) equipped with NTA software (version 3.1; Malvern Panalytical Ltd., Malverin, UK).

EV concentration and size were analyzed using a NanoSight NS300 (Malvern Panalytical, Malvern, UK) equipped with a 488 nm laser. EV suspensions were diluted (1:200–1:400) in filtered PBS. Samples were analyzed under constant flow conditions (flow rate: 30) at 25 °C and were captured with a camera level of 13–14 using NanoSight NTA software version 3.4 (Malvern Pana- lytical). Five independent measurements (60 s each) were obtained for each sample. Data are reported as mean ± standard error of mean (SE).

As previously described (Karkowska-Kuleta et al., 2020 (link)), to observe EVs, a JEOL JEM-2100 HT transmission electron microscope (JEOL, Tokyo, Japan) and negative stain transmission electron microscopy were used with formvar-coated, 300-mesh copper grids prepared for each EV sample using 2% uranyl acetate (Chemapol, Prague, Czech Republic). The size and concentration of EVs were measured as described in a previous work (Karkowska-Kuleta et al., 2020 (link)), using the nanoparticle tracking analysis (NTA) and NanoSight NS300 system with camera type sCMOS, laser Blue488, and NTA software Version 3.4 (Malvern Instruments, Malvern, UK).

The size distribution and concentration of exosomes were determined by nanoparticle tracking analysis (NTA) using the NanoSight NS300 system (Malvern Panalytical Ltd., Malvern, UK). The exosome samples were diluted 1000-fold in PBS for NTA measurements. The samples were infused with the syringe pump at a constant speed of 20 into the microfluidic flow cell equipped with a 53- nm laser and a high-sensitivity scientific CMOS camera. At least three videos per sample were recorded with the camera level of 11–13 for 30 s at 25 °C. All data were analyzed using NTA software (version 3.4; Malvern Panalytical Ltd., Malvern, UK) with a detection threshold of 5.

lEV concentration and size distribution were measured using a NanoSight NS300 (Malvern Panalytical, Malvern, UK) equipped with a 488 nm laser. lEV suspension was diluted (1:100–1:200) in filtered PBS. Samples were analyzed under constant flow conditions (flow rate: 30) at 25 °C, and were captured with a camera level of 13–14 using NTA software version 3.4 (Malvern Panalytical). Five independent measurements (60 s each) were obtained for each sample. Data are reported as mean ± standard deviation (SD).

The EV concentration (number of particles/mL) and size mode (nm) obtained with each technique, before and after magnetic bead immunocapture, was determined by dynamic light scattering using a NanoSight NS300 system (Malvern Panalytical, Worcestershire, UK).
To achieve a range of 10–100 particles per frame, 100 µL of each salivary EV isolate was diluted with freshly 0.22 µm-filtered 1X PBS to a final volume of 500 µL and loaded into the detection chamber with the NanoSight syringe pump accessory. NTA acquisition settings were maintained identical to characterize EVs obtained before and after magnetic bead immunocapture (blur size: 5 × 5, max jump distance: 13 pixels, minimum track length: 5 consecutive frames, threshold: 2). For each EV sample, five 90-s videos with a minimum of 200 valid tracks/video (minimum of 1000 valid tracks/sample) were recorded at room temperature. The camera level was manually adjusted to achieve optimal visualization of particles, with values ranging from 10 to 12 for each EV sample. Data were processed with the NTA software (version 3.4; Malvern Panalytical, Worcestershire, UK). The concentration of salivary EV isolates was adjusted for the dilution factor applied (1:5).

To characterize the size and particle concentration of Pf-EVs, the isolated EVs were diluted with sterile 0.22 µm-filtered PBS at the appropriate dilution to obtain the optimum measurement range. The diluted samples were injected into the Nanosight NS300 Analyzer (Malvern Pananalytical; Worcester, UK). Nanosight acquisition and analysis parameters were as follows: camera level: 14–15; detection threshold: 5; syringe pump speed: 50 µL/s; number of videos acquired per sample: 5; video duration: 60 s. Data analysis was performed using NTA software version 3.4 (Malvern Pananalytical; Worcester, UK). The system was washed with PBS between samples.