Subsequently, we tested CaspeR3 responsiveness in transiently transfected HeLa cells. Living cells were moni tored at 37 C with Leica SP2 confocal microscope. The fluorescence was evenly distributed in the cytosol and nucleus with no aggregation or non specific localization observed. Importantly, both green and red signals were reliably Sorafenib Tosylate IC50 stable upon blue exci tation in various irradiation conditions for hours. No reversible or irreversible fluorescence bleaching or photo conversion were observed. Apoptosis was induced by treatment with 2 M stau rosporine Approximately 40 50 min after staurosporine infusion, cells dem onstrated rapid and pronounced changes in green to red fluorescence signal ratio, indicating activa tion of caspase 3. Later these cells demonstrated character istic membrane blebbing.
The average contrast in living cells reached 3. 8 fold. FLIM One of the most powerful and quantitative Inhibitors,Modulators,Libraries approaches to measure FRET Inhibitors,Modulators,Libraries changes is fluorescence lifetime imaging, which measures the effect of the acceptor on the excited state lifetime of the donor. If the acceptor is in close proximity, the lifetime is reduced. The reduction of the fluorescence lifetime is a kinetic parameter that can be determined independently of probe concentration, microscope optical path and moderate levels of photob leaching. Therefore, the reduction of the donor lifetime is an extremely robust and quantitative estimate of FRET efficiency that is directly proportional to the amount of uncleaved substrate. We performed FLIM measurements of the non fused TagGFP and the TagGFP within the CaspeR3 sensor in living cells.
These experiments demon strated substantial differences in the detected fluorescence lifetimes. Accordingly, upon staurosporine induced Inhibitors,Modulators,Libraries apoptosis, fluorescence lifetime of TagGFP within CaspeR3 changed dramatically, switching from 1. 5 ns to 2. 5 ns. The FRET efficiency of the uncleaved CaspeR3 is among the highest measured by FLIM and compares favorably to a red to green caspase sensor reported previously, with a FRET efficiency of 25%. Since the FRET efficiency of the cleaved substrate is zero, the dynamic range of the sensor is rather high, indi cating that TagGFP TagRFP FRET pair will be an excellent tool for the high content FLIM based screenings on living cells. Conclusion Most reported FRET indicators are based on historically first CFP YFP pairs.
Inhibitors,Modulators,Libraries However, these FRET pairs are not really the most convenient and effective. Indeed, spec tral separation of overlapping cyan and yellow emission spectra can never Inhibitors,Modulators,Libraries be complete, and use of narrow band pass filters results in dramatic loss of emission. Besides, the relative high levels of autofluorescence in blue cyan region of visible spectrum and phototoxicity using near UV selleckchem excita tion further complicates their application. Reported FRET pairs containing red fluorescent acceptors suffer from tetramerization and demonstrate lower con trast.