Mannitol

Secretoneurin (SN) and Alexa Fluor® 647 labeled SN were synthesized and purified by piChem (Graz, Austria) and provided as freeze-dried product. Full length SN sequence was used as reported by Fischer-Colbire et al: TNE IVE EQY TPQ SLA TLE SVF QEL GKL TGP NNQ [30 (link)]. Single stranded DNA oligonucleotides (ODN 1826 control—5’-TCC ATG AGC TTC CTG AGC TT-3’) were obtained from InvivoGen (San Diego, USA) and contain full phosphorothioate backbone. ODN 1826 control does not cause any immunostimulatory effect and was therefore used as non-active ODN component. Protamine free base (Grade IV, Histone free) was purchased from Sigma Aldrich (Vienna, Austria). For our in-vivo biodistribution study oligonucleotides were synthesized by Biospring (Frankfurt am Main, Germany) with the same sequence and backbone properties as mentioned above.
Stock solutions for each component were prepared in Milli-Q (MQ) water and stored at -24°C. MQ water (ultra-pure water) was provided by a Milli-Q-Gradient system (Merck-Millipore, Darmstadt, Germany). In addition, the water was sterilized before usage at 121°C for 20 min.
For HPLC measurements, acetonitrile (HPLC gradient grade) was obtained from VWR (Leuven, Belgium) and trifluoroacetic acid from Merck (Darmstadt, Germany). Mannitol was purchased from Carl Roth (Vienna, Austria) and was used as cryoprotectant for lyophilization.

Activated charcoal, agar agar Kobe I, and mannitol were purchased from Carl Roth (Karlsruhe, Germany). Soy flour (Hensel Bio-Voll-Soja) was from W. Schoenenberger GmBH Co. KG (Magstadt, Germany). HPLC grade methanol was purchased from VWR International GmbH (Bruchsal, Germany). Ethyl acetate was distilled prior to use. For preparation of media, double distilled water was used. All other chemicals were from Sigma-Aldrich (Taufkirchen, Germany). Petri dishes were purchased from Greiner Bio-One GmbH (Frickenhausen, Germany).