Time-Resolved Fluorescence Resonance Energy Transfer [TR-FRET] Assays for Biochemical Processes.

Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is a fluorescence based technique which enables the analysis of molecular interactions in biochemical processes. Principle of TR-FRET is based on time-resolved fluorescence (TRF) measurement and fluorescence resonance energy transfer (FRET) between donor and acceptor molecules. To generate FRET signal, donor and acceptor molecules must show spectral overlap and should be in close proximity to each other and display suitable dipole orientation. The specific signal is acquired from molecules of interest via interactions of donor and acceptor molecules. TR-FRET technique is widely used for studying kinase assays, cellular signaling pathways, protein-protein interactions, DNA-protein interactions, and receptor-ligand binding. There are various propriety applications of TR-FRET. Two different sample protocols are summarized in this review.

Heparin toxicity in cell culture: a critical link in translation of basic science to clinical practice.

Heparin is a universal drug used frequently for its anticoagulant effects. The variabilities in distribution and tendency of heparin to accumulate in tissues cause increased tissue concentrations despite normal serum levels. We aimed to underline the toxic effects of heparin in cell culture make projections for clinical applications. L929 mouse fibroblastic cell line was plated in 96-well culture plates at an initial density of 5000 cells/well. Heparin was prepared in 10 different concentrations (10-300 units/well). Following 3 days of incubation, viabilities were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for each concentration in each day and compared. The viability of cells decreased significantly with increasing doses of heparin; at least 50 units/well in the first and second days and at least 20 units/well in the third day (P < 0.05 for each). There was statistically significant difference when the viabilities of cells treated with same heparin concentration in different days were compared (P < 0.05). The authors clearly demonstrated the toxic effects of heparin in cell culture, toxic effects increased as the dose increased. To prevent the unwanted clinical side-effects of heparin further studies should be made and more accurate testing methods should be developed to determine the effective tissue concentration of heparin.