Amicon centrifugal filter device

Approximately 200 µl of exosomes from the control groups were mixed and incubated with 1-µM DiR (Invitrogen, USA) for 30 min at room temperature. Free DiR was then removed from DiR-labeled exosomes by using Amicon centrifugal filter devices (Millipore, USA). After fish anesthesia with benzocaine, the DiR-labeled exosomes were injected into three healthy fish (average weight, 1.2 g) at the posterior dorsal aorta by a microinjection system (WPI PV820, USA) with a dose of 8 µl/fish. After 6 h, the fluorescence images of live fish body from both dorsal and ventral sides and major organs after dissection were recorded by IVIS (Wilber NEWTON 7.0, France).

The VWF levels secreted into the supernatant media and present in the cell lysates of the six healthy ECFCs (three different samples each; N = 18) and IP-ECFCs (six samples from various cell passages, the same as the passage numbers of the healthy individuals; N = 6) were determined. Seventy-two hours after seeding cells (at a density of 1.5 × 10⁶ cells/10 mL in 75 cm² flasks), the supernatant medium was collected, and cells were lysed. VWF present in media and cell lysates was concentrated on Amicon centrifugal filter devices (Millipore, Burlington, MA, USA), VWF:Ag levels were measured, and VWF multimers were analyzed by electrophoresis on 1.2% and 1.6% SDS-agarose gel [33 (link),64 (link)].

The Dia-EGFpET11d plasmid was transformed into Escherichia coli Rosetta DE pLysS (Novabiochem, Schwalbach, Germany) [14 (link)]. Cells were incubated overnight at 30 °C in Lysogeny Broth (LB) containing 100 mg/mL ampicillin. Cells were centrifuged (5 min, 3000 g) and then the pellet was dissolved in 2 L of LB containing 50 mg/mL ampicillin. Cells were grown overnight to a final optical density of 0.8. Isopropyl β-D-1-thiogalactopyranoside (IPTG, 2 mL, 1 M) was added for 3 h at 30 °C. Cell harvests were obtained by centrifugation (10 min, 5000 g, 4 °C). Cell pellets were resuspended in 20 mL phosphate-buffered saline (PBS, pH 7.4) at 20 °C. Fusion proteins were released from the FRENCH Press at 1500 psi after thawing. The solution was centrifuged (30 min, 4 °C) and adjusted with 10 mM imidazole. The construct applied to nickelnitrilotriacetate chromatography (Ni-NTA Agarose, Qiagen, Hilden, Germany). The fractions were analyzed by SDS-PAGE (12%) after elution with imidazole (20–250 mM). The fraction containing dianthin-EGF (DE) was concentrated in Amicon centrifugal filter devices (30 kDa Millipore, Eschborn, Germany). Imidazole was removed by buffer exchange (PBS) with PD10 columns (GE Healthcare, Munich, Germany). A BCA assay (Pierce, Rockford, IL, USA) was used to determine the protein concentration.

hBD-MSCs were passaged 5–6 times and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing a low glucose concentration supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% gentamycin. Then, hBD-MSCs were incubated with basal medium (BM, Gibco) with serum (conditioned medium; CM) and with 800 μg/ $\mathrm{m}\ell$ AGE-albumin (AA/CM) with or without 400 ng/ $\mathrm{m}\ell$ sRAGE (AA/CM/sRAGE) for two days. These medium were collected in a conical centrifuge tube and cell debris was discarded. Supernatants were concentrated by Amicon centrifugal filter devices (Millipore, USA) following the manufacture’s protocol.
Human umbilical vein endothelial cells (HUVECs; CEFObio, Seoul, Korea) were cultivated with EGM^TM-2 growth medium including bovine brain extract with heparin, human epithelial growth factor, hydrocortisone and GA-1000 (Lonza, Walkersville, MD) in a 1% gelatin (Sigma-Aldrich, St Louis, MO) coated culture dish. Murine macrophage cells (Raw 264.7) and rattus norvegicus skeletal muscle cells (L6) were also used in this study. These cells were purchased from Korean Cell Line Bank and cultured in DMEM with a high glucose concentration supplemented with 10% FBS and 100 U/ $\mathrm{m}\ell$ penicillin and 100 μg  $\mathrm{m}\ell$ streptomycin. All cells were maintained in a 5% CO₂ humidified incubator at 37 °C.