Malvern nanosight ns300 instrument

Extracellular vesicles were harvested from primary nHDFs at passage 5-7, from normal and transduced cells using a 15-day serum starvation method previously described (Walravens et al., 2021 (link)). Briefly, cells were grown to near confluence (∼90%) at 20% O₂/5% CO₂ at 37°C. Cell bed was washed 2x with warmed phosphate-buffered saline (PBS) and then incubated in IMDM without serum supplementation for 15 days in the same environment. Conditioned medium was collected, centrifuged at 3,000 × g for 10 min to remove dead cells and debris, then filtered through a 0.45-μm PES filter to remove apoptotic bodies and protein aggregates, and frozen for later use at −80°C. EVs were purified using centrifugal ultrafiltration with a 100-kDa molecular weight cutoff filter (Sigma-Millipore). EV preparations, before and after concentration were analyzed by NTA using the Malvern Nanosight NS300 Instrument (Malvern Instruments) with the following acquisition parameters: camera levels of 15, detection level less than or equal to 5, number of videos taken = 5, and video length of 30 s.

Exosomes were harvested from transduced fibroblasts at passage 16 under hypoxia conditioning. Briefly, cells were grown in 636 cm² CellStack chambers (Corning, Corning, NY, United States) until confluence. The flask was then washed three times with phenol-free IMDM and conditioned in the same for 15 days under 20% O₂. Conditioned media were collected, pooled, and filtered through 0.45 μm filter to remove apoptotic bodies and cell debris and frozen at −80°C for later use. Extracellular vesicles were isolated using centrifuge-based ultrafiltration with a molecular weight cutoff of 100 kDa (MilliporeSigma). Isolated EVs were quantified using Malvern Nanosight NS300 Instrument (Malvern Instruments, Malvern, UK) with the following acquisition parameters: camera levels of 15, detection level less than or equal to 5, number of videos taken 4, and video length of 30 s.