Nta 3.0 analytical software

Exosomes were isolated through standard centrifugation steps, as previously described [19 (link)]. Briefly, the culture medium was centrifuged at 300×g for 10 min to remove detached cells. The resulting solution was centrifuged at 2000×g for 20 min, followed by centrifugation at 10,000×g for 45 min. The supernatant was then filtered through a 0.22 μM filter mesh (Merck Millipore, USA). The filtrate was ultracentrifuged at 100,000×g for 70 min at 4 °C to form pellets containing exosomes. The resulting supernatant was carefully retrieved without disturbing the exosome-containing pellets. Exosome containing pellets were washed with a large volume of ice-cold particle-free PBS. A second round of ultracentrifugation was carried out under the same conditions (100,000×g for 70 min at 4 °C). The resulting exosome-containing pellets were resuspended in 100 μl of PBS. Exosomes were examined by electron microscopy using negative staining. Further, exosomes were quantified using a NanoSight NS300 instrument (Malvern Instruments Ltd. UK) equipped with NTA 3.0 analytical software (Malvern Instruments Ltd. UK). Protein concentration in exosomes was estimated using Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).

NTA was performed using the NanoSight NS300 system (Malvern Instruments, UK).^{65 (link)} The NanoSight polystyrene latex calibration beads, 100 and 200 nm, were applied to check the instrument performance. The size of the exosomes was determined based on both light scattering and Brownian motion and calculated using the Stokes–Einstein equation with NTA 3.0 analytical software (Malvern). NTA software was used to measure the concentration of the particles (particles/ml) and size distribution (in nm). Each sample was measured three times.

Exosomes were examined by electron microscopy using negative staining and quantified using the NanoSight NS300 instrument (Malvern Instruments Ltd. UK) equipped with NTA 3.0 analytical software (Malvern Instruments Ltd. UK).

After incubation in DMEM/F12 medium supplemented with 10% exosome-free FBS for 48 h, the medium was obtained. Exosome Precipitation Solution (System Biosciences, CA, USA) was applied to isolate the exosomes. The blood was collected in ethylenediaminetetraacetic acid (EDTA) containing collection tubes, and exoEasy Maxi Kit (Qiagen, Hilden, Germany) was used to isolate serum exosomes. The image of the exosomes was taken at 100 keV by using transmission electron microscopy (JEM-1-11 microscope, Tokyo, Japan). The Nanosight NS500 instrument (Malvern Instruments Ltd, Malvern, UK) and NTA 3.0 analytical software (Malvern Instruments Ltd) were used for quantification.

For exosome purification, CM was pre-cleared by filtration through a 0.22 μm PVDF filter (Millipore, USA). Exosomes were isolated from the CM by differential centrifugation steps as previously described [53 (link)]. The size and concentration of the exosomes were quantified using NanoSight NS300 instrument (Malvern Instruments Ltd., UK) equipped with NTA 3.0 analytical software (Malvern Instruments Ltd., UK). In addition, the plasma exosomes were isolated using ExoQuick Plasma prep and Exosome precipitation kit (SBI, USA). For exosomal RNA and protein extraction, exosomes were pretreated with RNase or Proteinase K, respectively. The exosome fraction protein content was assessed by Pierce® BCA Protein Assay kit (Thermo Scientific, USA).

Exosomes were isolated from FHC cell conditioned medium by 0.22 μm filtration and ultracentrifugation as we previously described [24 ]. Transmission electron microscopy (JEM-1-11 microscope, Japan) was used to image exosomes at 100 keV, and quantified by Nanosight NS300 instrument (Malvern Instruments Ltd. UK) equipped with NTA 3.0 analytical software (Malvern Instruments Ltd. UK).