Development of In‐Cell Western Assays Using Far‐Red Fluorophores

Nathan J. Moerke1, Gregory R. Hoffman2

1 Harvard Medical School, ICCB‐Longwood Screening Facility, Boston, Massachusetts, 2 Harvard Medical School, Department of Cell Biology, Boston, Massachusetts
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch100153
Online Posting Date:  March, 2011
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Abstract

The in‐cell western (ICW) technique is a cell‐based immunoassay method for quantitative measurement of protein expression or phosphorylation levels that can be used for both small molecule and siRNA screening. The method involves growth of cells in microplates, fixation, permeabilization, and staining with specific antibodies and/or cell labeling dyes. ICW assays take advantage of the properties of near‐infrared dyes to achieve higher signal‐to‐noise ratios than are possible for methods utilizing fluorophores in the visible range of the spectrum, and typically involve measurements using two fluorescent channels: one to measure levels of the target of interest, and one to measure total cell number for normalization. The ICW method is readily adaptable to high‐throughput format and has been successfully used with a variety of targets and cell lines. The protocols in this unit describe an ICW procedure for quantitative measurement of rpS6‐phosphorylation as an endpoint for monitoring mTORC1 signaling in HeLa cells. This assay can be used for small molecule or siRNA screening, and with modification is adaptable to other cell lines and targets. Curr. Protoc. Chem. Biol. 3:39‐52 © 2011 by John Wiley & Sons, Inc.

Keywords: in‐cell western; ICW; high‐throughput screening; mTOR; rpS6; rapamycin; phosphorylation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Compound Treatment of Cells for In‐Cell Western Assay
  • Basic Protocol 2: siRNA Transfection of Cells for In‐Cell Western Assays
  • Basic Protocol 3: In‐Cell Western Assay for Quantification of rpS6 Phosphorylation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Compound Treatment of Cells for In‐Cell Western Assay

  Materials
  • Dimethyl sulfoxide (DMSO)
  • Compounds being studied: e.g., Rapamycin, LY294002, and UO126 (Sigma)
  • Dishes of HeLa cells
  • Trypsin/EDTA (Invitrogen, cat. no. 25200‐056)
  • DMEM (CellGro, cat. no. 10‐013‐CV)
  • Fetal bovine serum (FBS) (PAA Laboratories, cat. no. A15‐301)
  • Penicillin/streptomycin (CellGro, cat. no. 30‐002‐CI)
  • Tissue culture hood
  • Polypropylene 384‐well compound storage plates (Thermo Scientific, cat. no. AB‐1056 )
  • Pin transfer apparatus (V&P Scientific, http://www.vp‐scientific.com) (Rudnicki and Johnston, )
  • Matrix WellMate (or other automated liquid dispenser for multiwell plates) (Rudnicki and Johnston, )
  • WellMate manifold (sterile)
  • Black opaque 384‐well tissue culture microplates (Corning, cat. no. 3712)
  • Standard tissue culture incubator capable of maintaining an environment of 37°C and 5% CO 2

Basic Protocol 2: siRNA Transfection of Cells for In‐Cell Western Assays

  Materials
  • Non‐targeting control, PDK1, S6K1, S6K2, mTOR, and PLK1 siRNA pools (Dharmacon)
  • Dish containing HeLa cells
  • DMEM (CellGro, cat. no. 10‐013‐CV)
  • Fetal bovine serum (FBS; PAA Laboratories, cat. no. A15‐301)
  • Oligofectamine transfection reagent (Invitrogen, cat. no. 12252‐011)
  • Opti‐MEM (Invitrogen, cat. no. 11058‐021)
  • Penicillin/streptomycin (CellGro, cat. no. 30‐002‐CI)
  • Tissue culture hood
  • 384‐well siRNA storage plates (Eppendorf, cat. no. 951020745)
  • Matrix WellMate (or other automated liquid dispenser for multiwell plates) (Rudnicki and Johnston, )
  • WellMate manifold (sterile)
  • Black opaque 384‐well tissue culture microplates (Corning, cat. no. 3712)
  • Centrifuge capable of holding multiwall plates
  • 20°C incubator
  • Liquid handling robot (e.g., Velocity 11 Bravo) (Rudnicki and Johnston, )
  • Standard tissue culture incubator capable of maintaining an environment of 37°C and 5% CO 2

Basic Protocol 3: In‐Cell Western Assay for Quantification of rpS6 Phosphorylation

  Materials
  • Phosphate‐buffered saline (PBS; see recipe)
  • EM‐grade formaldehyde (Polysciences, cat. no. 04018)
  • Plates to be assayed (see protocol 1 or protocol 2)
  • Triton washing solution (see recipe)
  • Alexa Fluor 680 succinimidyl ester (see recipe)
  • Blocking solution (see recipe)
  • Anti‐rpS6 phospho‐S235/S236 antibody (Cell Signaling Technologies, cat. no. 2211)
  • IRDye 800CW‐conjugated goat anti‐rabbit secondary antibody (LI‐COR Biosciences, cat. no. 926‐32211)
  • 24‐pin aspiration wand (Drummond) modified by slipping cut pieces of laboratory tubing over the ends of the wand so that the pins remain a few millimeters above the bottom of the microplate wells when aspirating
  • Matrix WellMate manifold (nonsterile; Thermo Scientific)
  • Matrix WellMate microplate dispenser (or other automated liquid dispenser for multiwell plates; Thermo Scientific) (Rudnicki and Johnston, )
  • Multiwell plate washer (e.g., Bio‐Tek ELx405) (Rudnicki and Johnston, )
  • Dark box or cupboard or aluminum foil
  • Parafilm
  • Metal foil seals for microplates (Corning, cat. no. 6570)
  • Near‐infrared‐capable plate scanner (e.g., Aerius, LI‐COR)
  • Spreadsheet or graphing software
  • Curve fitting software (optional)
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Figures

Videos

Literature Cited

Literature Cited
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   Hoffman, G.R., Moerke, N.J., Hsia, M., Shamu, C.E., and Blenis, J. 2010. A high‐throughput, cell‐based screening method for siRNA and small molecule inhibitors of mTORC1 signaling using the In Cell Western technique. Assay Drug Dev. Technol. 8:186‐199.
   Inoki, K., Corradetti, M.N., and Guan, K.L. 2005. Dysregulation of the TSC‐mTOR pathway in human disease. Nat. Genet. 37:19‐24.
   Olive, D.M. 2004. Quantitative methods for the analysis of protein phosphorylation in drug development. Exp. Rev. Proteomics 1:327‐341.
   Rudnicki, S. and Johnston, S. 2009. Overview of liquid handling instrumentation for high‐throughput screening applications. Curr. Protoc. Chem. Biol. 1:43‐54.
   Stockwell, B.R., Haggarty, S.J., and Schreiber, S.L. 1999. High‐throughput screening of small molecules in miniaturized mammalian cell‐based assays involving post‐translational modifications. Chem. Biol. 6:71‐83.
   Tsui, M., Xie, T., Orth, J.D., Carpenter, A.E., Rudnicki, S., Kim, S., Shamu, C.E., and Mitchison, T.J. 2009. An intermittent live cell imaging screen for siRNA enhancers and suppressors of a kinesin‐5 inhibitor. PLoSOne 4:e7339.
   Wong, S.K. 2004. A 384‐well cell‐based phospho‐ERK assay for dopamine D2 and D3 receptors. Anal. Biochem. 333:265‐272.
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   Zhang, J.H., Chung, T.D., and Oldenburg, K.R. 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4:67‐73.
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