8m guanidine hcl

TRIS, Formic Acid, and Acetonitrile were obtained from Merck KGaA. Hydrochloric Acid 37% was obtained VWR BDH code 20255.290. 8M Guanidine HCl was obtained from ThermoFisher Scientific code 24115. N‐Glycanase was produced from Agilent formerly Prozyme code GKE‐5006D. Trypsin/Lys‐C mixture was produced from Promega Mass Spec Grade code V5071. Ultrafiltration devices Amicon Ultra‐4 10K were produced from by Merck Millipore code UFC801096. Purified water was obtained by a Milli Q System Millipore mod. Advantage A10 produced from Merck Millipore. Maleimide was produced from Merck Sigma code I29585. Acquity BEH UPLC C18 2.1 x 100 mm cod. 186002352 from Waters. Formic Acid cod 100264 from Merck. Acetonitrile cod. 1.00029 from Merck.

The presence of the following cytokines and growth factors, known to contribute to cell recruitment, in the int-hAM and dev-hAM CM was tested from three pooled donors: endothelial growth factor (EGF); vascular endothelial growth factor (VEGF); basic fibroblast growth factor (bFGF); platelet-derived growth factor (PDGF)-BB; interleukin (IL)-6; and IL-8.
The presence of IL-6, and IL-8 in the int-hAM and dev-hAM CM was investigated using a multiplex chemiluminescent array (Aushon Biosystems, Billerica, MA). For VEGF and PDGF-BB testing, 250 μL CM was incubated with 250 μL 8 M guanidine HCl (Thermo Fisher Scientific, Waltham, MA) containing protease inhibitor (Roche, Basel, Switzerland) at 4°C overnight with rotation. Samples were centrifuged at 300 g for 10 min at 4°C and then desalted using Zeba Desalting Columns (Thermo Fisher Scientific) as per the manufacturer's instructions. Desalted samples were collected and stored at −80°C until further testing by enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) as per the manufacturer's instructions. The guanidine HCl treatment of samples before measurement of VEGF and PDGF-BB was performed to eliminate factors that interfere with VEGF and PDGF-BB detection by ELISA. The presence of EGF and bFGF in the CM was also tested by ELISA as per the manufacturer's instructions (R&D Systems).