I administered Ang-1 as 50ng/ml for a kinetic assay for 1hr in HUVEC (passage 9) in Collagen-1 (200ug/ml) coated well, but the fluorescence (in arbitrary unit) was very high indeed. Almost 1000 at the 5th min. Previously it was about 200-300. 

What could affect mostly to give such high intensity? 

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