Caspases,Paracaspases, and Metacaspases Methods and Protocols

135

4. Place the oocytes in a 50-mL conical tube containing OR-2
   buffer. Wash the oocytes several times with OR-2 buffer,
   removing as much blood and extraneous tissue as possible.


5. Incubate the oocytes with agitation in OR-2 buffer and 2.8 U
   of Liberase blendzyme for ~2 h at room temperature.


6. When completely digested ( see Note 21 ), wash the oocytes
   several more times with OR-2 buffer, pouring off as much
   debris and tissue as possible, leaving only oocytes behind.


7. Place the oocytes in a glass petri dish containing OR-2 buffer
   supplemented with 10 % fetal bovine serum (FBS) and 0.2 %
   gentamicin, and store at 18 °C.


8. Using a dissecting (stereo) microscope, sort as many stage VI
   oocytes as are required for the experiment. Stage VI oocytes
   have a very distinctive appearance, very similar to mature, laid
   eggs ( see Fig. 4 ). A characteristic that can help differentiate stage
   VI oocytes is a “white band” around the midline of the oocyte.
   Store stage VI oocytes in OR-2 buffer supplemented with FBS
   and gentamicin at 18 °C until ready to use ( see Note 22 ).

The large size and physical robustness of X. laevis oocytes makes

them very amenable to study and microinjection. This means

studying caspase activation directly in intact oocytes is also possi-

ble. The principle of the technique is similar to that used to study

caspase-3/7 activity in egg extract; utilizing a fl uorogenic, near-

infrared caspase-3 substrate (LI-COR). Upon induction of apop-

tosis and activation of caspase-3, this substrate fl uoresces, displaying

a peak emission at 789 nm. While this technique does require more

3.11 Assessment
of Caspase-3/7
Activity in Intact
X. laevis Oocytes
Using a Near-Infrared
Caspase Substrate

Fig. 4 Healthy stage VI oocytes, in comparison to stage VI oocytes undergoing apoptosis. Healthy stage VI X.
laevis oocytes were harvested from mature, female frogs as described in Subheading 3.10. Oocytes were
untreated as control ( left panel ) or treated with trans-dehydroandrosterone (DHEA) ( right panel ), incubated at
room temperature, and monitored for induction of apoptosis. Note the distinctive “white dot” on the oocytes
undergoing apoptosis in the right panel ( black arrowheads ; McCoy and Nutt, unpublished data)

Methods for the Study of Caspase Activation in the Xenopus laevis Oocyte...