Nanosight lm10 system

Small EVs were obtained from cell supernatants, as previously described with some modifications [24 (link),27 (link),69 (link)]. In brief, cells were incubated in DME medium with ultracentrifuged 5% FBS for 48 hours. The supernatants were collected and centrifuged at 300 g for 5 min and then at 2000× g for 10 min. The supernatant was then centrifuged at 10,000× g for 30 min, followed by filtration through a 0.2-μm pore filter (17597K, Sartorius, Gottingen, Lower Saxony, Germany). The collected supernatant was then subjected to preparation procedures by either ultracentrifugation at 100,000× g for 70 min as previously described [24 (link),25 (link)] or an affinity-based method using MagCapture (Fujifilm Wako Chemicals, Tokyo, Japan) [70 (link)] for small EVs isolation. Nanoparticle tracking analysis were performed using a NanoSight LM10 system (Malvern Panalytical, Westborough, MA, USA). The amount of dsDNA in EVs was determined by quantitative Real-Time PCR using these primers: human LINE1, 5′-CAAACACCGCATATTCTCACTCA-3′ (forward), and 5′-CTTCCTGTGTCCATGTGATCTCA-3′ (reverse) [23 (link),24 (link)].

The OMV size, volume, surface area, and count distributions were obtained on a NanoSight LM10 system (Malvern Instruments Ltd., Worcestershire, UK) using Nanoparticle Tracking and Analysis (NTA) 3.2 software. All samples were diluted 1:1000 in PBS or CHES with camera shutter and gain optimized for data collection. Three 90 s videos were recorded, and frame sequences were analyzed under auto particle detection and tracking parameters, including detection threshold, pixel blur, minimum track length, and minimum expected particle size. All samples were run at room temperature (RT).

To measure the concentration and size distribution of isolated EVs we used a NanoSight LM10 system (Malvern Panalytical, Malvern, UK). In brief, we diluted EV samples 1:20 with filtered (0.22 μm) 1× PBS to achieve the instrument’s optimal particles per frame range. At the end of each measurement, the pump was washed several times with filtered 1× PBS to avoid cross-sample contamination. The camera settings were adjusted per the manufacturer’s instructions, samples were recorded in three repeats of 20 s, and data were analyzed using the NanoSight NTA 2.3 software (Malvern Panalytical, Westborough, USA).

The hydrodynamic diameter of the siRNA complexes was determined by DLS, using a Zetasizer APS (Malvern Panalytical, Spectris plc, Egham, Surrey, UK) with a nominal 5 mV He-Ne laser operating at a 633 nm wavelength. The refractive index of the lipid-based carriers and polymer-based carriers was 1.33 and 1.450, respectively, and the viscosity was 0.8872 cP at 25 °C. For each sample, three separate measurements were conducted with 20 runs each. The size of the lipidoid and commercial complexes was analyzed by NTA using a NanoSight LM10 system (Malvern Panalytical, Spectris plc, Egham, Surrey, UK) fitted with a high-sensitivity cCMOS camera and a 405 nm laser. Each sample was appropriately diluted in PBS before the measurement. Videos of 60 s were recorded and analyzed with the NTA software (version 3.1, build 3.1.45). The concentration ranges of the particles were between 10⁸ and 10⁹ particles per mL. Three measurements per sample were taken.

The size distribution and particle number/concentration of exosomes were measured by nanoparticle tracking analysis (NTA) using a Nanosight LM10 system with blue (488 nm) laser (Malvern Instruments, UK). Exosomes were diluted in filtered deionised water to obtain 20–60 vesicles per field of view for optimal tracking. Three videos of 30 s were taken and analysed using the NanoSight NTA 3.2 software. The area under the histogram for each triplicate measurement was averaged and used as one particle concentration measurement. All NTA measurements were done with identical system settings for consistency.

Isolation of exosomes from adipose tissues was performed following the methods described by Wei et al. (17 (link)) and Yu et al. (18 (link)). Epididymal white adipose tissue (eWAT), visceral white adipose tissue (vWAT), and subcutaneous white adipose tissue (sWAT) from mice were excised and cut into small pieces (5 mm × 5 mm). The shredded tissue was cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% exofree-fetal bovine serum (FBS) (2 mL media/g adipose tissue), and the culture supernatant was collected after 48 h. The supernatant was then centrifuged at 500 × g for 5 min, then 2,500 × g for 15 min, at a temperature of 4°C to remove large debris and dead cells. Exosomes were separated from the resulting supernatant using Exoquick-TC™ precipitation reagent (System Biosciences, Mountain View, CA, United States), per the manufacturer’s instructions. The resulting exosome pellet was resuspended in PBS for application purposes. The identification and quantification of the exosomes were performed using a NanoSight LM10 system (Malvern Panalytical Ltd., Malvern, UK), while the exosomal protein content was evaluated using the Bradford protein assay.

The numbers and sizes of particles in each fraction were estimated by nanoparticle tracking analyses (NTA) using the NanoSight LM10 system (Malvern Instruments, Worcestershire, UK), as previously describe^{20 (link),81 (link)}. In brief, silica beads with a diameter of 150 nm (24320, Polysciences, PA, USA) were used as a calibration for the 10K fraction, whereas 100 nm diameter beads (24041, Polysciences) were used for the 160K fraction and supernatant. The camera level (CL) and detection threshold (DT) were set at values of CL 12/DT 10 and CL 14/DT 4. The samples were diluted with PBS filtered through a 0.1 µm syringe filter (6789-1301, GE Healthcare UK Ltd., Buckinghamshire, UK) to reach the desired concentration between 2 × 10⁸ and 1 × 10⁹ particles/mL. This concentration was based on silica beads, with a known concentration to correct for unevenness. For each sample, 30 s captures per sample were recorded, and each measurement was independently performed five times. NTA software version 2.3 (Malvern Instruments) was used for data analyses. An average histogram was plotted from the data of five measurements.