Cell‐Based Assays Using the Fluorometric Imaging Plate Reader (FLIPR)

Kristi L. Whiteaker1, James P. Sullivan1, Murali Gopalakrishnan1

1 Abbott Laboratories, Abbott Park, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 9.2
DOI:  10.1002/0471141755.ph0902s09
Online Posting Date:  May, 2001
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Abstract

A critical component of the drug discovery and development process is the identification of novel pharmacophores. Such discovery efforts are, in general, facilitated by rapid and high‐throughput cell‐based assays of receptor/ion channel‐mediated signaling processes to screen diverse chemical libraries of compounds possessing activator or inhibitor activities at the desired target. The availability of the Flurometric Image Plate Reader (FLIPR) has made rapid assays of cellular signaling processes feasible by simultaneous kinetics measurement of cell‐based fluorescence changes in a 96‐ or 384‐well format. This unit describes the application of the FLIPR in cell‐based kinetic assays for measuring membrane potential changes and intracellular calcium dynamics.

     
 
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Table of Contents

  • Basic Protocol 1: FLIPR Measurement of Membrane Potential Changes in Cultured Cells Using bis‐(1,3‐Dibutylbarbituric Acid)Trimethine Oxonol [DiBAC4(3)]
  • Basic Protocol 2: Measurement of Intracellular Calcium Changes in Human Medulloblastoma TE671 Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Figures

Videos

Literature Cited

   Arndts, D. 1999. Ca2+ kinetic registrations by FLIPR in electrically stimulated cell culture. Abstr. 3rd Int. Cell Analysis Products Users Meet. Monterey, Calif.
   Bencherif, M. and Lukas, R.J. 1991. Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line. J Pharmacol Exp Ther. 257:946‐953.
   Di Virgilio, F., Steinberg, T.H., and Silverstein, S.C.. 1990 Inhibition of fura‐2 sequestration and secretion with organic anion transport blockers. Cell Calcium. 11:57‐62.
   Dickinson, K.E., Baska, R.A., Cohen, R.B., Bryson, C.C., Smith, M.A., Schroeder, K., and Lodge, N.J. 1998. Identification of [3H]P1075 binding sites and P1075‐activated K+ currents in ovine choroid plexus cells. Eur. J. Pharmacol. 345:97‐101.
   Epps, D.E., Wolfe, M., and Groppi, V. 1994. Characterization of the stead‐state and dynamic fluorescence properties of the potential‐sensitive dye bis‐(1,3‐dibutylbarbituric acid)trimethine oxonol (Dibac4(3)) in model systems and cells. Chem Phy Lipid. 69:137‐150.
   Fadayel, G.M., Izzo, N.J., and Bahinski, A. 1999. High‐throughput optical measurement of membrane potential:Expression and block of a voltage‐dependent potassium channel. Abstr. 3rd Int. Cell Analysis Products Users Meet. Monterey, Calif.
   González, J.E. and Tsien, R.Y. 1995. Voltage sensing by fluorescence resonance energy transfer in single cells. Biophys J. 69:1272‐1280.
   González, J.E. and Tsien, R.Y. 1997. Improved indicators for cell membrane potential that use fluroscence resonance energy transfer. Chem Biol. 4:269‐277.
   González, J.E., Oades, K., Leychkis, Y., Harootunian, A., and Negulescu, P.A. 1999. Cell‐based assays and instrumentation for screening ion‐channel targets. Drug Discov. Today. 4(9):431‐439.
   Gopalakrishnan, M., Whiteaker, K.L., Molinari, E.J., Davis‐Taber, R.A., Monteggia, L.M., Scott, V.E.S., Buckner, S., Milicic, I., Cain, J., Postl, S., Brioni, J.D., and Sullivan, J.P. 1999. Characterization of ATP‐sensitive potassium channels (KATP) in guinea‐pig bladder smooth muscle cells. J. Pharmacol. Exp. Ther 289:551‐558.
   Groebe, D., Gopalakrishnan, S.M., Hahn, H., Warrior, U., Traphagen, L., and Burns, D. 1999. Use of a Fluorometric Imaging Plate Reader in high throughput screening. SPIE Proc. 3603:277‐306.
   Jorgensen, T.D. 1999. Monitoring Ca2+‐activated K+ channels using FLIPR. Abstr. 3rd Int. Cell Analysis Products Users Meet. Monterey, Calif.
   Kao, J.P.Y., Harootunian, A.T., and Tsien, R.Y. 1989. Photochemically generated cytosolic calcium pulsed and their detection by Fluo‐3. J. Biol. Chem. 264:8179.
   Kuntzweiler, T.A., Arneric, S.P., and Donnelly‐Roberts, D.L. 1998. Rapid assessment of ligand actions with nicotinic acetylcholine receptors using calcium dynamics and FLIPR. Drug Dev. Res. 44:14‐20.
   Miller, T., Molinari, E., Anderson, K.L., Davis‐Taber, R., Scott, V., Brioni, J.D., Sullivan, J.P., and Gopalakrishnan, M. 1999a Pharmacological and molecular characterization of ATP‐sensitive K+ channels in the TE671 human medulloblastoma cell line. Eur. J. Pharmacol. 370:179‐185.
   Miller, T.R., Witte, D.G., Ireland, L.M., Kang, C.‐H., Roch, J. M., Masters, J.N., Esbenshade, T. A., and Hancock, A. A. 1999b. Analysis of apparent noncompetitive responses to competitive H1‐ histamine receptor antagonists in Fluorescent Imaging Plate reader‐based calcium assays. J. Biomol. Screen 4:249‐258.
   Rock, D. 1999. Using intracellular Ca2+ measurements to evaluate activity of Na+ channels in cultured neurons using the FLIPRTM system. Abstr. 3rd Int. Cell Analysis Products Users Meet., Monterey, Calif.
   Schroeder, K.S. and Neagle, B.D. 1996. FLIPR: A new instrument for accurate, high throughput optical screening. J Biomol Screen. 1:75‐81.
   Sherf, B.A., Navarro, S.L., Hannah, R.R., and Wood, K.V. 1996. Dual‐Luciferase Reporter Assay, an advanced co‐reporter technology integrating firefly and renilla luciferase assays. Promega Notes #57.
   Simchowitz, L. and Bibb, J.A. 1990 Functional analysis of the modes of anion transport in neutrophils and HL‐60 cells. Annu Rev. Physiol. 52:381‐397.
   Sirotina‐Meisher, A., Stauruch, M.J.O., and Boltz., R.C.D. 1999. Drug screening measurement of membrane potential in human T cells using the FLIPR system. Abstr. 3rd Int. Cell Analysis Products Users Meet. Monterey, Calif.
   Whiteaker, K.L., Davis‐Taber, R., Molinari, E.J., Hoogenboom, L., Cain, J., Scott, V.E.S., Sullivan, J.P., Brioni, J.D., and Gopalakrishnan, M. 1999. Cardiac and vascular KATP channels: molecular and pharmacologic distinctions. FASEB J., 816.4.
Key References
   Annual International Cell Analysis Products Users Meeting (1990) (organized by Molecular Devices, Sunnyvale, Calif). Abstracts.
   Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene, OR.
   FLIPR (Fluorometric Imaging Plate Reader User Manual), Molecular Devices, Sunnyvale, CA.
Internet Resources
  http://www.moldev.com
  Molecular Devices Web site. Information/updates of FLIPR, protocol notes on FLIPR‐based applications; other cell analysis products.
   http://www.probes.com
  Molecular Probes Web site. An excellent site for information of various fluorescence applications and dyes.
   http://www.atcc.org
  Web site of American Type Culture Collection (ATCC). Comprehensive supplier of cell lines.
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