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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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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Basic Protocol 1: Direct Measurement of Intracellular Pressure

  • 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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Literature Cited

Literature Cited
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  Fox, J.R. and Wiederhielm, C.A. 1973. Characteristics of the servo‐controlled micropipet pressure system. Microvascular Res. 5:324‐335.
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  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.
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