A High‐Content Assay for Biosensor Validation and for Examining Stimuli that Affect Biosensor Activity

Scott D. Slattery1, Klaus M. Hahn1

1 Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.15
DOI:  10.1002/0471143030.cb1415s65
Online Posting Date:  December, 2014
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Abstract

Biosensors are valuable tools used to monitor many different protein behaviors in vivo. Demand for new biosensors is high, but their development and characterization can be difficult. During biosensor design, it is necessary to evaluate the effects of different biosensor structures on specificity, brightness, and fluorescence responses. By co‐expressing the biosensor with upstream proteins that either stimulate or inhibit the activity reported by the biosensor, one can determine the difference between the biosensor's maximally activated and inactivated state, and examine response to specific proteins. We describe here a method for biosensor validation in a 96‐well plate format using an automated microscope. This protocol produces dose‐response curves, enables efficient examination of many parameters, and unlike cell suspension assays, allows visual inspection (e.g., for cell health and biosensor or regulator localization). Optimization of single‐chain and dual‐chain Rho GTPase biosensors is addressed, but the assay is applicable to any biosensor that can be expressed or otherwise loaded in adherent cells. The assay can also be used for purposes other than biosensor validation, using a well‐characterized biosensor as a readout for effects of upstream molecules. © 2014 by John Wiley & Sons, Inc.

Keywords: biosensor; HC screening; microplate assay; fluorescence; FRET

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Transfection of LinXE or HEK 293T Cells with Gradients of Biosensor and/or Regulators in 96‐Well Microplates
  • Basic Protocol 2: Automated Microscopy of Cells Transfected with Biosensor in a 96‐Well Microplate
  • Basic Protocol 3: Analysis Algorithm for Biosensor Response in Microplate Image Data
  • Support Protocol 1: Matlab Scripts for Analysis of Biosensor Response in Microplate Image Data
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Transfection of LinXE or HEK 293T Cells with Gradients of Biosensor and/or Regulators in 96‐Well Microplates

  Materials
  • 100 μg/ml poly‐L‐lysine (PLL; mol. wt. 150,000 to 300,000; Sigma‐Aldrich, cat. no. P4832)
  • Calcium‐ and magnesium‐free Dulbecco's phosphate‐buffered saline (CMF‐DPBS; appendix 2A)
  • LinXE or HEK 293T cells near confluency in 75‐cm2 (T‐75) flask
  • 0.25% trypsin/2.21 mM EDTA (Corning Cellgro, cat. no. 25‐053‐CI)
  • Complete DMEM: Dulbecco's modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and no antibiotics
  • TE buffer ( appendix 2A)
  • Dulbecco's modified Eagle Medium (DMEM), serum‐ and antibiotic‐free
  • DNAs to be transfected (see Strategic Planning and Fig.  ; biosensor and expression plasmids can be custom synthesized; various mammalian expression vectors may be used; the example shown here uses pTriEx‐4 as a vector, which is available from Addgene; expression constructs for Rac1 dual‐chain biosensor, donor and acceptor controls, Vav2(DH/PH), and RhoGDI‐1, are available from the Hahn Lab):
    • Empty vector DNA
    • Biosensor expression plasmid
    • Regulator expression plasmid
    • Donor‐only fluorescent protein expression plasmid
    • Acceptor‐only fluorescent protein expression plasmid
    • Solution #1 consists of only empty vector DNA for a mock transfection
    • Solutions #2 and #3 consist of donor‐only and acceptor‐only expression plasmids, respectively, plus empty vector
    • Solution #4 consists of Biosensor expression plasmid, plus empty vector
    • Solution #5 consists of Biosensor and Regulator expression plasmids.
  • Lipofectamine transfection reagent (Life Technologies)
  • Dulbecco's modified Eagle Medium (DMEM) containing 20% fetal bovine serum (FBS) and no antibiotics
  • Single well V‐bottom liquid reservoirs that fit multichannel pipettor
  • 12‐channel pipettor for 20 to 200 μl
  • Black walled, tissue culture treated MicroClear 96‐well plate (Greiner Bio‐One, cat. no. 655090)
  • Multichannel aspirator (8 or 12 channels)
  • Clear 96‐well V‐bottom sterile polystyrene microplates (Greiner Bio‐One, cat. no. 651161)
  • Additional reagents and equipment for standard cell culture techniques including trypsinization and counting cells (unit 1.1) and imaging ( protocol 2)

Basic Protocol 2: Automated Microscopy of Cells Transfected with Biosensor in a 96‐Well Microplate

  Materials
  • Imaging medium (see recipe)
  • Microplate with cells to be imaged ( protocol 1)
  • Automated microscope with the following features:
    • A motorized stage that is capable of precise movements in x, y, and z, and that accepts microplates
    • Laser‐based or image‐based autofocus.
    • Motorized filter and dichroic mirror wheels containing filters and dichroic mirrors capable of capturing images in the following channels:
    • Donor channel: excitation of the donor and collection of the donor emission without collection of any acceptor emission
    • FRET channel: excitation of the donor with collection of the acceptor emission
    • Acceptor channel: excitation of the acceptor and collection of the acceptor emission, without any excitation of the donor
    • Software that allows programming the automated imaging of an entire 96‐well plate
  • 12‐channel pipettor for 20 to 200 μl

Basic Protocol 3: Analysis Algorithm for Biosensor Response in Microplate Image Data

  Materials
  • Microplate image data ( protocol 2)
  • A desktop computer running any software capable of batch processing of images, such as Matlab, and a spreadsheet program

Support Protocol 1: Matlab Scripts for Analysis of Biosensor Response in Microplate Image Data

  Materials
  • Microplate image data ( protocol 2)
  • A personal computer equipped with Matlab software. We have tested the functions using Matlab R2013a (8.1.0.604). The functions may also work on earlier or later versions.
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Figures

Videos

Literature Cited

Literature Cited
  Garcia‐Mata, R., Boulter, E., and Burridge, K. 2011. The ‘invisible hand’: Regulation of RHO GTPases by RHOGDIs. Nat. Rev. Mol. Cell Biol. 12:493‐504.
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  Itoh, R.E., Kurokawa, K., Ohba, Y., Yoshizaki, H., Mochizuki, N., and Matsuda, M. 2002. Activation of Rac and Cdc42 video imaged by fluorescent resonance energy transfer‐based single‐molecule probes in the membrane of living cells. Mol. Cell. Biol. 22:6582‐6591.
  Lakowicz, J.R. 1999. Principles of Fluorescence Spectroscopy, Second Edition. Kluwer Academic/Plenum Publishers, New York, N.Y.
  Kraynov, V.S., Chamberlain, C., Bokoch, G.M., Schwartz, M.A., Slabaugh, S., and Hahn, K.M. 2000. Localized Rac Activation dynamics visualized in living cells. Science 290:333‐337.
  Machacek, M., Hodgson, L., Welch, C., Elliott, H., Pertz, O., Nalbant, P., Abell, A., Johnson, G.L., Hahn, K.M., and Danuser, G. 2009. Coordination of Rho GTPase activities during cell protrusion. Nature 461:99‐103.
  Miyawaki, A. and Tsien, R.Y. 2000. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. In Methods in Enzymology, Vol. 327: Application of Chimeras in Cell Physiology (J. Thorner, S. Emr, J. Abelson, and M. Simon, eds.) pp. 472‐500. Academic Press, New York.
  Moon, S.Y. and Zheng, Y. 2003. Rho GTPase‐activating proteins in cell regulation. Trends Cell Biol. 13:13‐22.
  Pertz, O., Hodgson, L., Klemke, R., and Hahn, K.M. 2006. Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440:1069‐1072.
  Rossman, K.L., Der, C.J., and Sondek, J. 2005. GEF means go: Turning on Rho GTPases with guanine nucleotide‐exchange factors. Nat. Rev. Mol. Cell Biol. 6:167‐180.
  Thompson, G., Owen, D., Chalk, P.A., and Lowe, P.N. 1998. Delineation of the Cdc42/Rac1‐binding domain of p21‐activated kinase. Biochemistry 37:7885‐7891.
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