Reaction tubes

All chemicals, cell culture media, and supplements were obtained from Sigma–Aldrich Chemie GmBH (Taufkirchen, Germany) or Carl Roth GmbH (Karlsruhe, Germany), except for fetal bovine serum (FBS), which was obtained from Thermo Fisher Scientific GmBH (Dreieich, Germany). Transwell plates and inserts were acquired from Corning (Amsterdam, Netherlands). Other cell culture materials, such as dishes, flasks, and reaction tubes were bought from Sarstedt (Nuembrecht, Germany). Min-U-Sil5 particles were purchased from U.S.Silica (Katy, TX, USA). Endotoxin content was assessed with the LAL Chromogenic Endotoxin Quantitation Kit (ThermoFisher Scientific, Karlsruhe, Germany). Primers for RT-qPCR were synthesized by Eurofins (Ebersberg, Germany). PCR reagents were purchased from Macherey–Nagel (Dueren, Germany), Applied Biosystems (Foster City, WI, USA), Teknova (Hollister, CA, USA), Fluidigm (San Francisco, CA, USA), Bio-Rad (Munich, Germany) and New England Biolabs (Frankfurt am Main, Germany).

Adipose tissue derived mesenchymal stem cells from 4 horses (mixed breeds and sexes, aged 15 ± 4.64 years) and 3 dogs (mixed breeds and sexes, aged 4.81 ± 3.51 years) were cultured to obtain nanoparticles as previously described. After obtaining 24 ml per donor of cell culture supernatant containing EVs, as described above, we divided it into 5 ml reaction tubes (Sarstedt, Germany), so that a total of 6 tubes with a volume of 4 ml from each donor were available for exposure to different storage conditions. These tubes were processed as follows: EDTA (Carl Roth, Germany) was added to obtain a final concentration of 5 mM (tube 1), TRE (Carl Roth, Germany) was added to three reaction tubes to obtain a final concentration of 25 µM (tube 2), 50 µM (tube 3) or 250 µM (tube 4), one tube as control of fresh (tube 5) and one tube as frozen samples (tube 6) without addition of TRE or EDTA. The size and concentration of EVs in the fresh reaction tubes (tube 5) were measured immediately after EVs purification by nanoparticle tracking analysis (NTA), whereas all other preparations were frozen at − 20 °C for 8 days (tubes 1–4, 6). After the freezing period, the reaction tubes were thawed in a 37 °C water bath and subsequently measured by NTA.

Before any treatment blood samples used for all following measurements were collected from the cephalic vein and depending on the following analysis, blood was put in ethylenediaminetetraacetic acid (EDTA)-prepared tubes or serum separator tubes (both from Sarstedt AG & Co., KG, Nümbrecht, Germany). An analysis of haematology was conducted with a ProCyte Dx Analyzer (IDEXX GmbH, Kornwestheim, Germany), and an analysis of serum chemistry was conducted with a Thermo Scientific Konelab 20i Clinical Chemistry Analyzer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Blood for α-Klotho measurements was collected into serum separator tubes (Sarstedt AG & Co., KG, Nümbrecht, Germany), allowed to clot at room temperature, and then centrifuged at 1000× g for 20 min. Afterwards, serum was pipetted into reaction tubes (Sarstedt AG & Co., KG, Nümbrecht, Germany) and stored in aliquots at −80 °C until analysis.