Analytical Cytology: Applications to Neurotoxicology

Michelle C. Catlin1, Marina Guizzetti1, Rafael A. Ponce1, Lucio G. Costa1, Terrance J. Kavanagh1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.5
DOI:  10.1002/0471140856.tx1205s04
Online Posting Date:  May, 2001
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Abstract

This unit describes methods for analyzing the effects of neurotoxicants on cell cycle regulation by dual parameter flow cytometry and on cell signaling by quantifying intracellular calcium concentrations within individual cells by scanning confocal laser microscopy or using the fluorescent calcium probe fluo‐3.

     
 
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Table of Contents

  • Basic Protocol 1: Cell‐cycle Analysis of Glial Cells by BrdU/Hoechst Flow Cytometry
  • Support Protocol 1: Preparation of Primary Rat Astrocytes
  • Basic Protocol 2: Analysis of Intracellular Calcium in Individual Glial Cells Using Indo‐1/AM
  • Support Protocol 2: Generation of Calcium Calibration Curve
  • Alternate Protocol 1: Analysis of Intracellular Calcium in Individual Glial Cells Using Fluo‐3/AM
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Cell‐cycle Analysis of Glial Cells by BrdU/Hoechst Flow Cytometry

  Materials
  • Monolayer cultures of primary rat astrocytes (see protocol 2)
  • Complete DMEM medium/5% and 10% (v/v) FBS (see recipe)
  • Ca2+, Mg2+‐free Dulbecco's phosphate‐buffered saline (CMF‐DPBS; appendix 2A)
  • Serum‐free medium (see recipe)
  • Agent to be tested for mitogenic potential
  • 15 mM BrdU (see recipe)
  • Hoechst buffer (see recipe)
  • 100× ethidium bromide (see recipe)
  • 0.1% (v/v) trypsin in CMF‐DPBS
  • 24‐well tissue culture plates
  • 12 × 75–mm polystyrene tubes
  • Flow cytometer equipped with UV‐excitation (351 to 362 nm lines of argon‐ion laser, or 365 nm line of a mercury arc lamp)
  • MPLUS AV program (Phoenix Flow Systems) or equivalent flow cytometry software program for data analysis
  • Additional reagents and equipment for tissue culture and trypsinization of cells ( appendix 3B) and flow cytometry (unit 2.5)

Support Protocol 1: Preparation of Primary Rat Astrocytes

  Materials
  • Day‐21 rat fetuses
  • Complete DMEM medium/10% FBS (see recipe)
  • 75‐cm2 tissue culture flasks, poly‐D‐lysine coated
  • Additional reagents and equipment for tissue culture ( appendix 3B)

Basic Protocol 2: Analysis of Intracellular Calcium in Individual Glial Cells Using Indo‐1/AM

  Materials
  • 132 1N1 astrocytoma cells (available from Dr. J. Heller‐Brown, University of California at San Diego)
  • Complete DMEM medium/5% FBS (see recipe)
  • Serum‐free medium (see recipe)
  • Toxicant of interest
  • Krebs' bicarbonate buffer (see recipe)
  • Loading buffer (see recipe)
  • 2 µM Indo‐1/AM in recipeloading buffer
  • Stimuli (e.g., serum, mitogens, glutamate)
  • 2‐well coverglass chamber slides (Nunc)
  • Inverted, scanning confocal microscope equipped with a mercury arc lamp or argon ion laser capable of an emission wavelength of 351 to 363 nm
  • 445‐nm long‐pass dichroic filters (Omega Optical)
  • Neutral density filters (Omega Optical)
  • Two photomultiplier tubes or other fluorescent detectors (capable of quantifying fluorescence in the range of 400 to 410 nm and 500 to 530 nm simultaneously)
  • Data acquisition system for collecting and storing digital images
  • Spreadsheet software
  • Additional reagents and equipment for generation of a calcium calibration curve (see protocol 4)

Support Protocol 2: Generation of Calcium Calibration Curve

  • Calcium Calibration Buffer Kit 1 (Molecular Probes)
  • Ethanol
  • Indo‐1 free acid

Alternate Protocol 1: Analysis of Intracellular Calcium in Individual Glial Cells Using Fluo‐3/AM

  • 2 µM Fluo‐3/AM solution, in recipeloading buffer
  • 35‐mm plastic tissue culture dishes
  • Inverted, scanning confocal microscope equipped with mercury arc lamp or argon ion laser capable of emission wavelength of 488 nm
  • 530/20‐nm band pass filters
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Figures

Videos

Literature Cited

Literature Cited
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   Poenie, M. 1990. Alteration of intracellular Fura‐2 fluorescence by viscosity: A simple correction. Cell Calcium 11:85‐91.
   Ponce, R.A., Kavanagh, T.J., Mottet, N.K., Whittaker, S.G., and Faustman, E.M. 1994. Effects of methyl mercury on the cell cycle of primary rat CNS cells in vitro. Toxical col. Appl. Pharmacol. 127:83‐90.
   Poot, M., Hoehn, H., Kubbies, M., Grossmann, A., Chen, Y.C., and Rabinovitch, P.S. 1990. Cell cycle analysis using continuous bromodeoxyuridine labeling and Hoechst 33258‐ethidium bromide bivariate flow cytometry. Methods Cell Biol. 33:185‐198.
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Key References
   Rabinovitch and June, 1990. See above.
   Describes parameters for loading calcium‐sensitive dyes and their detection.
   Rabinovitch et al. 1988. See above.
  Describes rationale and technique for BrdU/Hoechst cell cycle analysis
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