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Measurement of [Ca2+] Using the Fluorometric Imaging Plate Reader (FLIPR)
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-FLIPR, a New Regulator of Death Receptor-induced Apoptosis
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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FLIPR: A New Instrument for Accurate, High Throughput Optical Screening
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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