Vivaspin 6 centrifugal concentrator

For the determination of the emulsifier content in the aqueous phase of the emulsion, the aqueous phase was separated by a self-developed centrifugation technique and analyzed refractometrically afterwards. Vivaspin^® 6 centrifugal concentrators with a cut-off of 300 kDa (Sartorius, Göttingen, Germany) were applied for separation. All centrifugation steps were performed at 22 °C with 500 g in an Allegra 64R centrifuge (Beckman Coulter, Krefeld, Germany). Before centrifugation of the emulsions, the Vivaspins^® were prepared in three washing steps. In each step, 5 mL of bidistilled water was centrifuged for 10 min to remove production-related residues of glycerol and sodium azide from the filter membrane, as these would interfere with the subsequent analytical procedure. To avoid dilution effects, a 10 min centrifugation step in dry condition was added to remove water residues from the membrane. Afterwards, 5 mL of emulsion was filled into the Vivaspins^® and centrifuged for 60 min, to achieve the separated aqueous phase as filtrate.
The emulsifier content in the filtrate was determined refractometrically in an Abbemat-WR refractometer (Anton Paar, Ostfildern-Scharnhausen, Germany). The refractive index of each filtrate was measured three times at a temperature of 25 °C. The evaluation was performed based on calibration lines.

frTet (4-(1,2,3,4-tetrazin-3-yl) phenylalanine) was purchased from Aldlab Chemicals (Woburn, MA, USA). TCO-Cy3 was purchased from AAT Bioquest (Sunnyvale, CA, USA). TCO-PEG4-maleimide (TCO-PEG₄-MAL) and amine-axially substituted TCO (TCO-amine) were purchased from FutureChem (Seoul, Korea). Pentafluorophenyl ester (PFP)-PEG₄-APN was obtained from CONJU-PROBE (San Diego, CA, USA). Disposable PD-10 desalting columns and Superdex 200 10/300 GL Increase columns were purchased from Cytiva (Uppsala, Sweden). Vivaspin 6 centrifugal concentrators with a molecular weight cut-off (MWCO) of 10 and 100 kDa were purchased from Sartorius (Göttingen, Germany). HSA and all other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise mentioned.

UMR106 cells were cultured as described and treated with 100 ng/mL myostatin for 24 h. The cell culture supernatant was stored at − 80 °C. C-terminal FGF23 was determined by an ELISA kit (Mouse/Rat FGF-23 (C-Term), Immuntopics, San Clemente, CA, USA) according to the manufacturer’s protocol. Intact FGF23 was determined in the supernatant after its concentration by means of Sartorius Vivaspin 6 Centrifugal Concentrators (Sartorius, Göttingen, Germany), with an ELISA kit (Mouse/Rat FGF-23 (Intact), Immuntopics).

BM-MSCs or AMF stimulated BM-MSCs (incubated overnight with 1 µg/ml of rAMF in DMEM) were lysed with 150 mM NaCl, 20 mM Tris–HCl, pH 7.4, 0.1% SDS, 1.0% Nonidet P-40, 0.5% Na-deoxycholate, 0.2 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail. Lysates were centrifuged at 12,000× g for 20 minutes and the supernatants were used as total cell lysates. CCM and TCM were 100-fold concentrated using Vivaspin 6 centrifugal concentrator (Sartorius-Stedim Biotech). Protein concentration was determined by Bradford protein assay (Bio-Rad). Protein was separated using SDS-PAGE and transferred onto nitrocellulose membrane (Hybond-ECL, Amersham Biosciences). Blots were blocked and incubated with anti-AMF polyclonal antibody (1∶700, sc-33777, Santa Cruz Biotechnology) and anti-AMFR (1∶500, AP2162a, ABGENT) at 4°C overnight. Finally, blots were incubated with the corresponding HRP-conjugated at room temperature for 1 hour. Reactions were visualized by chemiluminescence. Staining with colloidal Coomassie was performed as loading control for conditioned medium as previously reported [22] (link). Density of each band was quantified with Scion Image software (Scion Corporation, Frederick, MD).

Cell culture supernatant comprising the recombinant protein was harvested every second day and stabilized with 10 mM Tris–HCl, pH 8.0, 10 mM EDTA, 10 mM ascorbic acid, and 0.02% sodium azide. Ultracentrifugation was performed at 7800 rcf for 15 min followed by 40,000 rcf for 45 min at 4 °C. The clear supernatant was microfiltered (0.22 μm, polyethersulfone) and concentrated to 50 ml using a Sartorius Vivacell 250 PES Centrifugal Concentrator (Sartorius) with a molecular weight cutoff of 100 kDa and a pressure of 4 bar. Buffer was exchanged five times with 200 ml of 100 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and concentrated to a final volume of 10 ml. The concentrate was centrifuged at 21,000 rcf for 30 min, and recombinant protein was purified from the supernatant with Strep-Tactin sepharose (IBA) according to the manufacturer’s protocol. Elution fractions containing TconTS1 were pooled, and buffer was exchanged four times with 5 ml of 10 mM potassium phosphate buffer (pH 7.4) using a Vivaspin 6 Centrifugal Concentrator (Sartorius) at 2000 rcf and 4 °C for 20 min. Protein samples were stored at 4 °C. TconTS1 concentrations were determined with a Pierce BCA Protein Assay kit (Thermo Fisher Scientific) using bovine serum albumin as standard.