Despite the fact that epithelial oBRB and endothelial BBB have developed as separate entities with many site-specific functions, their transport and permeation characteristics display surprising similarities, that include the polarized expression of the two major efflux pumps P-gp and MRP.
We have developed a microarray-based system for cell adhesion profiling of large panels of cell-adhesive proteins to increase the throughput of in vitro cell adhesion assays, which are currently primarily performed in multiwell plates. Miniaturizing cell adhesion assays to an array format required the development of protocols for the reproducible microspotting of extracellular matrix (ECM) protein solutions and for the handling of cell suspensions during the assay. We generated ECM protein microarrays with high reproducibility in microspot protein content using nitrocellulose-coated glass microslides, combined with piezoelectric microspotting of protein solutions. Protocols were developed that allowed us to use 5000 cells or fewer on an array of 4 x 4 mm consisting of 64 microspots. Using this microarray system, we identified differences of adhesive properties of three cell lines to 14 different ECM proteins. Furthermore, the sensitivity and accuracy of the assays were increased using microarrays with ranges of ECM protein amounts. This microarray system will be particularly useful for extensive comparative cell adhesion profiling studies when only low amounts of adhesive substrate and cells, such as stem cells or cells from biopsies, are available.
A caspase sensor based on Förster resonance energy transfer between fluorescent proteins is reported. Enhanced cyan fluorescent protein anchored to the inner leaflet of the plasma membrane of living cells is optically excited by an evanescent electromagnetic field and transfers its excitation energy via a spacer (DEVD) to an enhanced yellow fluorescent protein. Upon apoptosis, DEVD is cleaved and energy transfer is disrupted, as proven by pronounced changes in fluorescence spectra and decay times. Fluorescence spectroscopy and lifetime imaging (FLIM) is combined with total internal reflection fluorescence microscopy (TIRFM) for selective detection of this membrane-bound caspase sensor. Fluorophores of the cytoplasm are thus excluded, and the signal-to-background ratio is increased considerably. In comparison with conventional or laser scanning microscopy, this permits long-term observation of apoptosis in live cell cultures using very low absorption and avoiding light-induced damages of the samples. '
2008International Society for Advancement of Cytometry Key terms caspase sensor; apoptosis; Förster resonance energy transfer; fluorescence lifetime microscopy; total internal reflection microscopy; live cell microscopy THE measurement of caspase activities is commonly used to detect and investigate programmed cell death. Cyan fluorescent protein (CFP) fused to yellow fluorescent protein (YFP) by a caspase-sensitive amino acid peptide (DEVD) has been developed as an indicator of caspase-3 activity in living cells (1,2). The peptide linker between CFP and YFP is short enough to bring the two fluorescent proteins in close proximity to each other resulting in Förster resonance fluorescence energy transfer [FRET; (3)]. Upon induction of apoptosis, FRET is interrupted due to cleavage of the linker by caspase-3, and thus loss of FRET is a direct indicator of caspase activity. The sensitivity of this genetically encoded caspase sensor has been improved by optimizing the amino acid sequence flanking DEVD for higher FRET efficiency and thus improving the dynamic range of signal generation (4,5). Caspase sensor constructs following this principle have since been used to investigate caspase activity in living cells. For example, the rate of apoptosis of whole cell populations was determined (6-8), or the temporally distinct onset of apoptosis in single cells within whole cell collectives was detected (8). Furthermore, cleavage sequences specific for different caspases were used to reveal the activation of these caspases in the same cell (9,10).Detection of events of such dynamic occurrence often requires a temporal and spatial resolution in data acquisition that are only obtainable by real-time or timelapse recordings. So far, changes in FRET of caspase sensor probes have been mostly detected by calculating the ratio of acceptor (YFP) and donor (CFP) emission using conventional standard fluorescence or confocal laser scanning microscopy (4,11). However, both microscopic methods can be harmful to living cells because...
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