Caspases,Paracaspases, and Metacaspases Methods and Protocols

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1. Mature female primed frogs.


2. 10 % benzocaine.


3. Dissection equipment.


4. Liberase blendzyme 3 (Roche, Switzerland) or similar.


5. OR-2 buffer: 5 mM HEPES pH 7.5, 82.5 mM NaCl, and
   2 mM KCl.


6. Fetal bovine serum.


7. Gentamicin.


8. Glass petri dish.


9. Low temperature incubator.


10. Dissecting (stereo) microscope.


11. Stage VI oocytes ( see Subheading 2.10 ).


12. OR-2 buffer ( see Subheading 2.10 ).


13. Black 96-well plate.


14. Model P-97 fl aming/brown micropipette puller (Sutter
    Instrument Co.) or equivalent.


15. Glass capillary tubing: borosilicate glass, 10 cm length, OD
    1.0 mm, ID 0.5 mm.


16. Ficoll PM 400.


17. Clean, glass microscope slide.


18. 40× MMR ( see Subheading 2.1 for recipe).


19. Microinjection apparatus.


20. Dissecting (stereo) microscope.


21. IRDye 800CW/QC-1 CSP-3 (LI-COR).


22. Recombinant cytochrome c ( Equine , Sigma-Aldrich).


23. Odyssey Infrared Imaging system (LI-COR) or equivalent.

3 Methods

The X. laevis egg/oocyte system provides a quick high- throughput

model for studying the biochemical events of apoptosis and caspase

activation. It recapitulates all the classical hallmarks of apoptosis

and caspase activation. Using the extract model system, it is also

possible to interrogate the intrinsic apoptotic pathway and dissect

each stage. The protocols described below will provide examples of

assessment of in vitro-translated caspase activation, how to deter-

mine if the initiating signal for induction of apoptosis is pre-

mitochondrial, assessing cytochrome c release from mitochondria,

assessment of executioner caspase (3/7) activation in the egg

2.9 Isolation
of Stage VI Oocytes
from Mature, Female
X. laevis Frogs

2.10 Assessment
of Caspase-3 Activity
in Intact X. laevis
Oocytes Using
Near-Infrared Caspase
Substrate

Francis McCoy et al.

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