Nanosight nta 2

Isolated EVs were analyzed for particle size and particle concentration with the NanoSight NS300 instrument (Malvern Panalytical, Ltd., Malvern, UK) using Nanosight NTA 2.3 software Malvern Panalytical, Ltd., Malvern, UK), as in [7 (link),19 (link),28 (link)]. Briefly, 2 μL of EVs was added to 998 μL of distilled (DI) water to prepare the NTA sample. The instrument was first flushed with approximately 3 mL of DI water. Approximately 300 μL of sample was loaded into the NanoSight NS300 for analysis.

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).

EV size and concentration were determined using nanoparticle tracking analysis on a NanoSight LM10 (Malvern Instruments, UK). 3 × 60 s videos per sample were taken (temperature: 25 °C; flow rate: 30 µL s⁻¹; camera type: sCMOS; laser type: Blue 405 nm; camera level: 10; 20–100 particles/frame) and analyzed using the Nanosight NTA 2.3 software (Malvern Instruments, UK) with a detection threshold of 5.^[53] EV morphology was determined using transmission electron microscopy (JEM1400; Jeol, USA) following negative staining with 1% uranyl acetate. Images were captured at 120 kV using an 1k CCD camera (Advanced Microscopy Techniques, Corp., USA).
Protein harvested from Caco‐2 cells and EVs (30 ng) was resolved by electrophoresis in reducing conditions, transferred to nitrocellulose membranes then probed with primary antibodies β‐catenin, (0.1 µg mL⁻¹; A1978; Sigma‐Aldrich, UK), ALIX (ALG‐2‐interacting protein; final concentration 1 µg mL⁻¹; ab24335; Abcam, UK), CD63 (0.5 µg mL⁻¹; ab199921; Abcam), or CANX (Calnexin; 1 µg mL⁻¹; A303‐695A; Bethyl Laboratories, USA) overnight at 4 °C. Secondary antibodies (1 h, room temperature) were IRDye 800CW conjugated anti‐rabbit (926‐32213) or mouse (926‐68073) IgG (0.2 µg mL⁻¹; Li‐Cor Biosciences, UK Ltd., UK). Blots were imaged on the Li‐Cor Odyssey Sa (Li‐Cor Biosciences, UK Ltd., UK).