Nta v2

SKOV3 (human ovarian carcinoma: ATCC^® HTB-77 TM) cells were cultured in RPMI-1640 medium (Invitrogen, USA) supplemented with 2 mM L-glutamine (Invitrogen, Paisley, UK), 10% FBS (PAA Laboratories, Leonding, Austria) and 1% penicillin-streptomycin (Invitrogen, Paisley, UK) at 37 °C and 5% CO₂. HEK-293 cells (human embryonic kidney: ATCC^® CRL-1573 TM) were grown in MEM (Invitrogen, USA) with 2 mM L-glutamine, 10% FBS, 1 mM sodium pyruvate and 1% penicillin-streptomycin (Invitrogen, USA) at 37 °C and 5% CO₂. Vectors (HAdV-5 luc and LacZ [β-galactosidase]; HAdV-5T* luc and LacZ containing the following mutations I421G, T423N, E424S, L426Y, E451Q in HVR7 and T270P and E271G in HVR5 showing reduced FX-binding capacity [8 (link)]) were propagated in HEK-293 cells and purified by CsCl gradient centrifugation [15 (link)]. Viral particles were determined by micro BCA assay (Life Technologies, Camarillo, CA, USA) using the formula 1 mg protein = 4 × 10⁹ virus particles (vp). Laser-based nanoparticle tracking analysis (NTA) was used to characterize the size of adenoviral particles from pure preparations with Nanosight NTA v2.3 software in a NanoSight LM14 (Malvern Panalytical, Malvern, UK). Plaque forming units (pfu)/mL were calculated by end-point dilution assay [15 (link)]. The luc vectors were used for all the in vitro experiments, whereas LacZ vectors were used for the in vivo analyses.

Nanoparticle tracking analysis (NTA) was used to determine particle size distribution and concentration of EV samples from RWPE1, CD9 low, CD151 high and WPE1-NB26 cell lines. Concentrated EV suspensions were diluted 1:1000 in PBS before introduction into the sample chamber of a Nanosight NS300 NTA system (Malvern, ATA Scientific, Taren Point, NSW, Australia) using a syringe pump (Harvard Apparatus, ATA Scientific) on a speed setting of 50. The NS300 was adjusted so that particles were in the focal plane and a scientific CMOS camera captured 3 × 60 s videos per sample. Post-acquisition analysis settings were kept constant between samples. Particle size distribution and concentration were determined using NTA v2.3 software (Malvern).