Analysis of Apoptosis Using Xenopus Egg Extracts

Sally Kornbluth1, Erica K. Evans1

1 Duke University Medical Center, Durham, North Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.12
DOI:  10.1002/0471143030.cb1112s09
Online Posting Date:  May, 2001
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Abstract

In the presence of a subcellular fraction enriched for mitochondria, after prolonged incubation the Xenopus egg extract can mimic biochemical aspects of apoptosis such as caspase activation, and DNA fragmentation. This unit describes preparation of an apoptotic extract from a crude interphase extract and an extract fractionated into latent and execution phases. An apoptotic extract can also be reconstituted from a fractionated interphase extract and purified mitochondria. Protocols are also included for monitoring apoptotic progression in the extract either by following activation of apoptotic proteases (caspases) or by assessing translocation of cytochrome c from the mitochondria to the cytosol.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Apoptotic Extracts and Assessing Apoptosis
  • Alternate Protocol 1: Separating Apoptosis into Latent and Execution Phases
  • Support Protocol 1: Measuring Capase 3–Like Activity
  • Support Protocol 2: Preparation of Mitochondria from Xenopus Egg Extracts
  • Support Protocol 3: Cytochrome c Release Assays
  • Support Protocol 4: Cytochrome c Release Assay using Purified Mitochondria and Cytosol
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Apoptotic Extracts and Assessing Apoptosis

  Materials
  • Crude interphase extract (unit 11.10)
  • 0.2 M phosphocreatine
  • 0.5 mg/ml creatine kinase
  • 0.2 M ATP
  • Hoechst 33258 (Bisbenzimide H 33258; Calbiochem)
  • Fluorescence microscope
  • Additional reagents and equipment for preparation of Xenopus crude interphase extract (unit 11.10)

Alternate Protocol 1: Separating Apoptosis into Latent and Execution Phases

  Materials
  • Freshly obtained Xenopus eggs, washed with egg lysis buffer (unit 11.10)
  • 0.2 M phosphocreatine
  • 0.5 mg/ml creatine kinase
  • 0.2 M ATP
  • 20× energy‐regenerating mix (unit 11.11)
  • Demembranated sperm chromatin (unit 11.10)
  • Hoechst 33258 (Bisbenzimide H 33258; Calbiochem)
  • 15‐ml polypropylene tubes
  • 5‐ml syringe and 18‐G needle
  • Additional reagents and equipment for preparing crude and fractionated interphase extracts (unit 11.10)

Support Protocol 1: Measuring Capase 3–Like Activity

  Materials
  • Assay buffer (see recipe), room temperature
  • Apoptotic egg extract (see protocol 1 or protocol 2)
  • Cleavage substrate: 2 mM Ac‐DEVD‐pNA (see recipe)
  • Cleavage inhibitor: 20× Ac‐DEVD‐CHO (see recipe)
  • 96‐well microtiter plate

Support Protocol 2: Preparation of Mitochondria from Xenopus Egg Extracts

  Materials
  • Fractionated interphase extract ( protocol 2 in unit 11.10)
  • Percoll solutions (see recipe)
  • MIB buffer (see recipe)
  • 2.5‐ml ultracentrifuge tubes (e.g., Beckman)
  • Beckman TL‐100 table‐top ultracentrifuge and TLS‐55 swinging‐bucket rotor
  • Additional reagents and equipment for preparing fractionated Xenopus interphase extract (unit 11.10)

Support Protocol 3: Cytochrome c Release Assays

  Materials
  • Crude extract for apoptotic reaction (see protocol 1)
  • 0.1‐µm ultrafree‐MC filters (Millipore)
  • Anti‐cytochrome c antibody (Pharmingen)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and immunoblotting (unit 6.2)

Support Protocol 4: Cytochrome c Release Assay using Purified Mitochondria and Cytosol

  Materials
  • Heavy membrane fraction containing mitochondria (see protocol 4)
  • Cytosolic fraction containing energy regenerating mix (unit 11.10)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and immunoblotting (unit 6.2)
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Figures

Literature Cited

Literature Cited
   Bellamy, C.O.C., Malcomson, R.D.G., Harrison, D.J., and Wyllie, A.H. 1995. Cell death in health and disease: The biology and regulation of apoptosis. Semin. Cancer Biol. 6:3‐16.
   Chao, D.T. and Korsmeyer, S.J. 1998. BCL‐2 family: Regulators of cell death. Annu. Rev. Immunol. 16:395‐419.
   Cosulich, S.C., Green, S., and Clarke, P.R. 1996. Bcl‐2 regulates activation of apoptotic proteases in a cell‐free system. Curr. Biol. 6:997‐1005.
   Evans, E.K., Kuwana, T., Strum, S.L., Smith, J.J., Newmeyer, D.D., and Kornbluth, S. 1997. Reaper‐induced apoptosis in a vertebrate system. Eur. Mol. Biol. Organ. J. 16:7372‐7381.
   Faure, S., Vignerson, S., Doree, M., and Morin, N. 1997. A member of the Ste/PAK family of protein kinases is involved in both arrest of Xenopus oocytes at G2/prophase of the first meiotic cell cycle and in prevention of apoptosis. Eur. Mol. Biol. Organ. J. 16:5560‐5561.
   Kuwana, T., Smith, J.J., Muzio, M., Dixit, V., Newmeyer, D.D., and Kornbluth, S. 1998. Apoptosis induction by caspase‐8 is amplified through the mitochondrial release of cytochrome c. J. Biol. Chem. 273:16589‐16594.
   Newmeyer, D.D., Farschon, D.M., and Reed, J.C. 1994. Cell‐free apoptosis in Xenopus egg extracts: Inhibition by Bcl‐2 and requirement for an organelle fraction enriched in mitochondria. Cell 79:353‐364.
   Villa, P., Kaufmann, S.H., and Earnshaw, W.C. 1997. Caspases and caspase inhibitors. Trends Biochem. Sci. 22:388‐393.
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