Agilent protein 230 kit

MSCs were lysed with 1% protease phosphatase inhibitor in RIPA extraction buffer and, total protein was measured using a Agilent Protein 230 kit (Agilent Technologies). Protein was loaded on precast 4–12% polyacrylamide Bis-Tris gels in a NuPage MOPS-buffer and SDS-PAGE western blotting was performed using a XCell SureLock™ Mini-Cell and a PowerEase 500 W power supply. For the transfer on nitrocellulose membranes with a 0.2 μm pore size a XCell II blot™ module was used (Invitrogen, Thermo Fisher Scientific). Membranes were subsequently blocked with non-fat dry milk (Biorad, Hercules, CA). A primary anti-IDO Ab (0.2 mg, clone H-11, Santa Cruz Biotechnology) was detected with HRP conjugate and Clarity Max Western ECL blotting substrate (Biorad) and the chemiluminescent signal was recorded with a Chemi-Doc documentation system (Biorad) for semi-quantification. For internal control, purified IDO protein (Bio-Techne, R&D Systems, Minneapolis, MN) was used at a total concentration of 25–200 ng.

The sample powders were solubilised using in 6M urea and 2% SDS buffer. All samples were diluted to 4 mg/mL (scales TE-124S-OCE, Sartorius AG, Gottingen, Germany), then shaken for 1 min by heavy duty vortex mixer VXHDDG (Ohaus, Parsippany, NJ, USA) at 2500 rpm. The suspensions were then shaken for 1.5 h by environmental shaker incubator ES-20 (Biosan, Ltd., Riga, Latvia) at room temperature. The suspensions passed 15 min centrifugation at G-force 2300 (centrifuge CM-6MT, Elmi Ltd., Riga, Latvia). The resulting supernatants of protein samples were collected and frozen.
Analyses were performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions using Agilent Bioanalyzer 2100 capillary electrophoresis system (Agilent, Santa Clara, CA, USA) with Agilent Protein 230 Kit (14–230 kDa sizing range). Briefly, aliquots of 4 µL unfrozen protein samples were mixed with 2 µL DDT denaturing solution, prepared according Agilent protocols (3.5 Vol, -% of 1M DTT), spined for 15 s and then heated at 95 °C for 5 min, cooled down and diluted to 90 µL with deionized water. Ladder, Gel-Dye mix and destaining solution were prepared and loaded according the Agilent assay protein protocols for Bioanalyzer 2100.

The amount of radio-labeled mannitol was quantified by mixing 0.5 mL of the withdrawn sample with 5 mL scintillation cocktail (Ultima Gold, Perkin Elmer, Waltham, MA, USA) and measured using liquid scintillation. FITC-dextran was quantified by fluorescence spectrophotometry (SpectraMax i3 x, Molecular Devises, San Jose, CA, USA) at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The marker solution diluted in the modified Krebs buffer was used as a standard. The amount of ovalbumin was determined using an Agilent Bioanalyzer 2100 with the Agilent Protein 230 Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. Briefly, a 40 µL sample was mixed with 2 µL dye and heated to 95 °C for 5 min. An amount of 6 µL of the mixture was loaded to the protein 230 chip after staining of the gel. The amount was quantitatively determined using a reference solution with the known concentration (800 ng/µL).