Direct Measurement of Intracellular Pressure

Ryan J. Petrie1, Hyun Koo2

1 Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, 2 Biofilm Research Laboratory and Department of Orthodontics, University of Pennsylvania School of Dental Medicine, Philadelphia
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 12.9
DOI:  10.1002/0471143030.cb1209s63
Online Posting Date:  June, 2014
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Abstract

A method to directly measure the intracellular pressure of adherent, migrating cells is described in this unit. This approach is based on the servo‐null method where a microelectrode is introduced into the cell to directly measure the physical pressure of the cytoplasm. We also describe the initial calibration of the microelectrode, as well as the application of the method to cells migrating inside three‐dimensional (3‐D) extracellular matrix (ECM). Curr. Protoc. Cell Biol. 63:12.9.1‐12.9.9. © 2014 by John Wiley & Sons, Inc.

Keywords: intracellular pressure; fibroblasts; motility; extracellular matrix

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Direct Measurement of Intracellular Pressure
  • Support Protocol 1: Calibrating the Microelectrode
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Direct Measurement of Intracellular Pressure

  Materials
  • Human primary dermal fibroblasts (unit 10.18)
  • High‐glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS)
  • Cell‐derived matrix (unit 10.19)
  • 1.7 mg/ml collagen gel (unit 10.18)
  • Sigmacote (Sigma, cat. no. SL2; optional)
  • Low‐walled glass‐bottom culture dish, 50/40 mm (Warner Instruments, cat. no. 64‐0760)
  • 50‐ml disposable conical tubes (e.g., BD Falcon)
  • Inverted microscope with environmental controls to maintain 37°C, 10% CO 2, and humidity
  • Reference electrode (World Precision Instruments, cat. no. DRIREF‐2)
  • 900A micropressure system (World Precision Instruments, cat. no. SYS‐900A)
  • 4‐axis motorized micromanipulator (Sutter Instrument, cat. no. MPC‐325)
  • Phase‐contrast microscope objective
  • 1.0‐mm outer diameter micropipet with filament, 0.5 µm opening (World Precision Instruments, cat. no. TIP05TW1F)
  • 2‐ml disposable plastic serological pipets
  • Microelectrode holder (1.0 mm) with Ag/AgCl half‐cell and air column connections (World Precision Instruments, cat. no. MEH6SF10)
  • Data acquisition system (World Precision Instruments, cat. no. LAB‐TRAX4‐24T)
  • BNC‐to‐BNC Cable, M‐M (World Precision Instruments, cat. no. 2851)
  • Computer with LabScribe 2 software (World Precision Instruments)
  • Additional reagents and equipment for growing cells in extracellular matrix (units 10.18 & 10.19)

Support Protocol 1: Calibrating the Microelectrode

  Additional Materials (also see protocol 1Basic Protocol)
  • 1 M KCl and 0.1 M KCl
  • MicroFil flexible needle (World Precision Instruments, cat. no. MF34G‐5)
  • Disposable 10‐ml syringe
  • Pressure calibration chamber (World Precision Instruments, cat. no. CAL900A)
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Figures

Videos

Literature Cited

Literature Cited
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  Cole, K.S. 1932. Surface forces of the Arbacia egg. J. Cell. Comp. Physiol. 1:1‐9.
  Dai, J. and Sheetz, M.P. 1999. Membrane tether formation from blebbing cells. Biophys. J. 77:3363‐3370.
  Fein, H. 1972. Microdimensional pressure measurements in electrolytes. J. App. Physiol. 32:560‐564.
  Fox, J.R. and Wiederhielm, C.A. 1973. Characteristics of the servo‐controlled micropipet pressure system. Microvascular Res. 5:324‐335.
  Kelly, S.M. and Macklem, P.T. 1991. Direct measurement of intracellular pressure. Am. J. Physiol. 260:C652‐C657.
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  Mitchison, J.M. and Swann, M.M. 1954. The mechanical properties of the cell surface: I. The cell elastimeter. J. Exp. Biol. 31:443‐460.
  Petrie, R.J. and Yamada, K.M. 2012. At the leading edge of three‐dimensional cell migration. J. Cell Sci. 125:5917‐5926.
  Petrie, R.J., Gavara, N., Chadwick, R.S., and Yamada, K.M. 2012. Nonpolarized signaling reveals two distinct modes of 3D cell migration. J. Cell Biol. 197:439‐455.
  Petrie, R.J., Koo, H., and Yamada, K.M. 2014. Generation of compartmentalized pressure by a nuclear piston drives 3D cell motility. Submitted for publication.
  Rabbany, S.Y., Funai, J.T., and Noordergraaf, A. 1994. Pressure generation in a contracting myocyte. Heart Vessels 9:169‐174.
  Rand, R.P. and Burton, A.C. 1964. Mechanical properties of the red cell membrane. I. Membrane stiffness and intracellular pressure. Biophys. J. 4:115‐135.
  Ridley, A.J. and Hall, A. 1992. The small GTP‐binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70:389‐399.
  Stewart, M.P., Helenius, J., Toyoda, Y., Ramanathan, S.P., Muller, D.J., and Hyman, A.A. 2011. Hydrostatic pressure and the actomyosin cortex drive mitotic cell rounding. Nature. 469:226‐230.
  Yanai, M., Kenyon, C.M., Butler, J.P., Macklem, P.T., and Kelly, S.M. 1996. Intracellular pressure is a motive force for cell motion in Amoeba proteus. Cell Motil. Cytoskel. 33:22‐29.
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