Data were evaluated using J 700 for Windows Standard Analysis software. MALDI TOF MS For detection of the VEGF A165 ATP complex, MALDI TOF MS was performed according to K?nig et al. with slight modifications. VEGF A165 and selleck compound its complexes were purified by reversed phase chromatography using C18 ZipTip pipet tips. Pipet tips were washed with elution solvent and equilibrated with aqueous sol vent before use. For purification, samples containing 27. 5 uM VEGF A165, optionally combined with ATP and MgCl2, were applied, rinsed with aqueous solvent and eluted into 5 uL of elution solvent. Purified samples were spotted onto a MALDI target coated with 0. 5 uL of 1% sinapinic acid in acetone. Subsequently, 0. 5 uL of 1% sinapinic acid in 40% acetonitrile was added. Spectra were obtained with MALDImicroMX.
Cell proliferation assay HUVECs were seeded in 96 well plates at 8 104 Inhibitors,Modulators,Libraries cells well containing 100 uL of Endothelial Cell Growth Medium with supplements. Seeded HUVECs were cultured Inhibitors,Modulators,Libraries under standard conditions CO2, 37 C for 24 h before EGM Inhibitors,Modulators,Libraries was replaced by 100 uL Endothelial Cell Basal Medium containing 0. 1% bovine serum albumin. After 1 h of incubation, media were replaced once more by 100 uL EBM containing VEGF A165 instead of BSA. In addition, Inhibitors,Modulators,Libraries AP was applied to selected samples at 40, 80 or 160 ng mL along with thegrowth factorand incubation was continued for 48 h. Subsequently, cell culture media were taken from separate samples treated in the same manner for measurement of extracellular ATP. Finally, proliferation of HUVECs was determined using the CellTiter 96 Aqueous One Solution Cell Pro liferation Assay kit according to the manufacturers instructions.
To that end, 20 uL of CellTiter 96 Aqueous One Solution was added to each well to be incubated for additional 3 h. The number of viable cells was directly proportional to the absorbance of a colored formazan product deter mined colorimetrically at Inhibitors,Modulators,Libraries 490 nm. Values are presented as means standard deviation. Luminometric measurement of extracellular ATP Extracellular ATP was determined in cell free samples of cell culture media employing the ATP Kit SL according to the manufacturers instructions. The bioluminescent reaction is based on the luciferase catalyzed oxidation of luciferin in the presence of oxy gen and ATP. Besides AMP, pyrophosphate and carbon dioxide, oxiluciferin is produced emitting light at 560 nm to be measured luminometrically. Light emission is proportional to the amount of ATP and sellckchem was measured in a two step procedure using an FB 12 Single tube Luminometer. First, the light intensity of the samples was quantified as relative light units second. Then, an internal ATP standard was added to each sample and light intensity was quantified again to give Ismp std.