Syringe filter

Once the cells were 80% confluent, they were first washed to ensure that nanoparticles in FCS did not interfere with the nanoparticles to be gained. For this, the medium in the cell culture flasks was replaced with 20 ml DMEM without FCS and the flasks were gently shaken on a shaker for 5 min. This washing procedure was repeated three times. Subsequently, 12 ml DMEM supplemented with 1% P/S and 1% insulin-transferrin-selenite solution (ITS-H, Capricorn, Germany) to compensate the absence of FCS was added to each cell culture flask. Three days later, the cell culture supernatant containing nanoparticles was harvested and centrifuged at 2700×g for 10 min at room temperature to remove cell debris. The supernatant was then filtered through a 0.2 µm syringe filter (83.1826.001, Sarstedt, Germany). The usual subsequent concentration process, e.g. ultracentrifugation, ultrafiltration or precipitation, was not performed in order to focus on the nanoparticles in the supernatant and the cryoprotectants without additional post-processing. Further purification and identification of nanoparticles containing equine small EVs, for example, has already been performed [29 (link)].
Since special attention was paid to short-term storage until additional concentration and/or experimental or even clinical application, no further processing of the nanoparticles obtained was carried out.

Reagents, chemicals, and solvents were from Sigma‐Aldrich (Merck), Apollo Scientific, or Thermo Fisher Scientific, except where stated. The HIF1α_556–574‐CODD (DLDLEMLAPYIPMDDDFQL), HIF2α_523–542‐CODD (ELDLETLAPYIPMDGEDFQL), and HIF3α_484–505‐CODD (ALDLEMLAPYISMDDDFQLN) and 3C cyclic (d‐YVWLTDTWVLSRTC)
²⁸ (link),
³⁵ (link) peptides were from GL Biochem (prepared with a C‐terminal amide). Water used for cell culture was purified using a Millipore Elix® 10 system (Merck Life Sciences) and autoclave sterilized (Crystal 300‐RP25, Rodwell Engineering Group). Water used for buffers and molecular experiments was filtered purified by a 0.22‐μm Milli‐Q filtration system (Milli‐Q, Merck Life Sciences). Kanamycin (final concentration 62 mM) was prepared in water sterilized by a benchtop autoclave (LTE TouchClave II, LTE Scientific; program: 121°C and 1 Bar for 15 min) and filtered for impurities with a 0.22‐μm syringe filter (Sarstedt).