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Ionized calcium plays a major role in the regulation of cellular processes in both eukaryotic and prokaryotic cells (1). A wide variety of cell surface receptors and ion channels utilize a calcium signal to initiate events such as cell motility, contraction, and secretion. To a large extent, the advent of fluorescent indicators of free calcium ion concentration that could be loaded into cells in a nondisruptive manner has been responsible for our current knowledge of cellular calcium homeostasis (2). Typically, the fluorescent signal has been monitored using cuvet-based fluorometers or by confocal fluorescent microscopy. Although acceptable for a number of applications, these methods are relatively labor intensive and are not suitable for screening large numbers of compounds.
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c-FLIPs (c-FLICE inhibitory proteins) play an essential role in regulation of death receptor-induced apoptosis. Multiple splice variants of c-FLIP have been described on the mRNA level; so far only two of them, c-FLIPL and c-FLIPS, had been found to be expressed at the protein level. In this report, we reveal the endogenous expression of a third isoform of c-FLIP. We demonstrate its presence in a number of T and B cell lines as well as in primary human T cells. We identified this isoform as c-FLIPR, a death effector domain-only splice variant previously identified on the mRNA level. Impor-/tantly, c-FLIPR is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex upon CD95 stimulation. Several properties of c-FLIPR are similar to c-FLIPS: both isoforms have a short half-life, a similar pattern of expression during activation of primary human T cells, and are strongly induced in T cells upon CD3/CD28 costimulation. Taken together, our data demonstrate endogenous expression of c-FLIPR and similar roles of c-FLIPR and c-FLIPS isoforms in death receptor-mediated apoptosis.
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Modern optical screening assays demand high data throughput along with uncompromised data fidelity. FLIPR (Fluorescent Imaging Plate Reader) was developed to perform quantitative optical screening for cell-based kinetic assays. FLIPR incorporates an integrated design, including low-level optical detection, precise temperature control, and precise fluid handling, all in one package. The unique aspect of FLIPR is that all 96 wells of a standard microplate are stimulated and optically measured simultaneously. Kinetic updates on all 96 wells can be obtained in under 1 sec, allowing for transient signals to be quantified. Demonstrated applications include measurements of intracellular calcium, intracellular pH, and membrane potential.
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