FLIPR® Calcium 3 Assay Kit: a new no-wash calcium flux assay for diversified receptor targets Abstract ID: 030527143240 Authors: David Donofrio, Zhiqiang Wang, and Jinfang Liao. Molecular Devices Corporation
Cell-based calcium flux assays are considered one of the most important screening techniques used in pharmaceutical drug discovery today. Due to variability in receptor expression, host cell characteristics and assay dynamics, receptor targets often need to be matched with specific calcium flux chemistries to achieve acceptable performance. We report here on the FLIPR Calcium 3 Assay Kit, a new "universal" fluorescence-based method for detecting changes in intracellular calcium concentration across a broad spectrum of biological targets. The Calcium 3 Assay Kit yields very high fluorescence emission intensity whereby strong signals are attained even in problematic targets that may previously have yielded weak signal intensity or no peak at all. The simple and reliable "mix-and-read" assay format substantially reduces, or eliminates entirely, the cell detachment and diminished response often associated with incubate-and-read wash procedures. Data is presented that compares signal intensity and well-to-well uniformity observed with the Calcium 3 Assay Kit and standard FLUO-3 AM and FLUO-4 AM dye incubation and wash procedures, as well as other mix-and-read kits.
Oxidative burst: Amplex Red detection of hydrogen peroxide release from activated human neutrophils and cell lines using FlexStation™ II384 microplate reader Abstract ID: 030530111841 Authors: Carole Crittenden*, Anna Christiensen, Molecular Devices Corporation; Elisabeth Gardiner, Kalypsis
The release of reactive oxygen species by activated human neutrophils plays an important role in the body’s defense against infection. This process of oxidative burst is correlated with many disease states, and is a primary component of phagocytosis and apoptosis in neutrophils. Here, we have activated human neutrophils differentiated HL-60 cells with phorbol 12-myristate 13-acetate (PMA) to undergo oxidative burst. In the presence of horseradish peroxidase, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) is oxidized by extracellular H2O2 to produce red-fluorescent resorufin. Capabilities for reagent addition, fluorescent signal detection, and data analysis by FlexStation II384 are used to optimize a cell-based kinetic medium throughput screen. Known inducers and suppressors of oxidative burst are used to compare the response of activated neutrophils and HL-60 cells in this system.
Microplate optimization in FLIPR Abstract ID: 030529130918 Authors: Jonathan Petersen and Carole Crittenden, Molecular Devices Corporation
FLIPR is a versatile instrument for screening kinetic cellular assays. In screening applications, it is important to optimize factors that can affect assay performance or reduce screening costs. Microplate choice is an important element in achieving suitable assay performance. Black-wall, clear bottom plates are routinely used in FLIPR assays. These plates generally give acceptable performance, but can be significantly more expensive than clear-wall microplates. In contrast, clear-wall, clear bottom plates are rarely used in FLIPR assays because of the perception that they will not give acceptable cross-talk performance. This poster will present a comparison of results obtained from assays conducted in black-wall, clear bottom plates with results obtained using less expensive clear-wall plates. The comparison will cover assays that use quench technology, which can reduce cross-talk, as well as wash assays. The plate performance will be compared in terms of Z-factor, cross-talk, and cost.
Screening compounds that modulate ionic currents using the IonWorks™ HT system Abstract ID: 030401104006 Authors: James Costantin, Andrew Wittel, and Wilhelm Lachnit, Molecular Devices Corporation
The IonWorks HT instrument is an automated high-throughput voltage clamp system that measures whole-cell currents from multiple cells in parallel using 384-well PatchPlates™. This system has the robustness required for single point screening of compounds that modulate ionic channel activity as well as the fidelity which allows IC50 determinations. For single point screening, data was generated from compound plates prepared containing randomly distributed compounds as well as controls. Following the identification of active wells, a 10-point IC50 curve for each compound was obtained. Results are presented for compounds that affect fast Na+ channels, and K+ channels including hERG and Kv1.5. The IC50 curves obtained are compared to the values obtained in parallel using the conventional patch clamp technique. This system provides an enormous opportunity to screen pharmaceutical compound libraries directly at the electrophysiological level and more effectively exploiting ion channel targets.
Expansion of IMAP® application: ATP tolerance and use of substrates with a high propoprtion of acidic residues Abstract ID: 030530120027 Authors: Annegret Boge, Liz Gaudet, Luke Lavis and Richard Sprotsman, Molecular Devices Corporation
IMAP is a homogenous, generic, FP based HTS system used to measure kinase, phosphatase and phosphodiesterase enzyme activity without the need for antibodies. The IMAP technology is based on the high affinity binding of phosphate by immobilized metal (MIII) coordination complexes on nanoparticles. In this study we show approaches to increasing the ATP tolerance of IMAP and to the use of peptide substrates with a high content of acidic amino acids. In this manner IMAP can be extended to include kinases that have been difficult to assay by this approach previously. Issues of type and placement of acidic amino acids in the substrate sequence are also discussed.
Adaptation of a high sensitivity 384-well solid phase assay platform to 1536 wells: CatchPoint™ for cyclic nucleotides and tyrosine kinase activity Abstract ID: 030530122619 Authors: Annegret Boge, Jonathan Petersen, Jeannie Nguyen, Gayle Teixeira and Richard Sportsman, Molecular Devices Corporation
The Molecular Devices CatchPoint platform is a very sensitive solid phase based assay system for assaying for cAMP, cGMP and tyrosine kinase activity. In order to make such a solid phase system amendable to ultra high HTS (even if it includes only one wash step) we have adapted it from 384-well plates to 1536-well plates. This adaptation was possible by the concurrent development of a new Washer/Dispenser System (AquaMax™ DW4) by Molecular Devices. We compare several performance parameters of the different assays between 384-well plates and 1536-well plates including sensitivity and variability.
Optimizing IMAP® for use with cell lysates and low volume microplates Abstract ID: TBD Authors: Yan Zhang, Molecular Devices Corporation
