Multichannel Systems HiClamp Manual - Manual (Page 79)

Appendix
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11.3.4 Collagenase Digestion and Defolliculation
Ovarian tissue contains immature and mature oocytes, as well as connective tissue from which
the oocytes must be separated. Ovarian tissue should be removed completely by collagenase
digestion. Oocytes are enveloped in a follicle cell layer which can cause trouble when plating
oocytes into well plates. Remaining pieces of follicular tissue causes oocytes to stick to the walls.
Oocytes will not move into correct positions in the middle of a well by themselves. Additionally,
the follicular cell layer sometimes hampers the impalement by the injection needle causing
damage to the oocytes or clogging of the tip. Therefore, it is important that isolated oocytes
are completely free of the surrounding follicular cell layer.
The whole procedure should be completed after about 2 to 2.5 hours. Please adjust the
collagenase concentration if this is not the case.
1. Divide the tissue with a razor blade and a forceps into smaller, approximately 0.5 cm
2
large
pieces.
2. Transfer the clumps into 50 ml Falcon tubes containing 40 ml of 1.5 to 2.0 mg/ml collagenase in
Barth’s without Ca 2+ . A volume of up to 7.5 ml of tissue can be put into a single tube. For more
tissue, use additional tubes. Otherwise, the collagenase digestion would take too much time.
3. Place the tubes onto the shaker and let them shake gently at 20 °C. Check the progress after
90 min (and then every 15 min), and vigorously shake the tube briefly by hand to accelerate
the process.
4. Typically 120 minutes after beginning of the treatment, practically all oocytes should be
isolated and the majority of them should have already lost their follicular cell layer. If not,
continue the collagenase treatment for a maximum of 30 min.
5. Wash the oocytes extensively with Barth’s solution (minimum of 5 times with 30 ml) to remove
the collagenase completely.
6. Then fill up the tube (approx. to 45 ml) and put it back onto the shaker for 10 minutes.
7. Change the solution to Barth’s without Ca 2+ and put it onto the shaker again for another
10 minutes. Practically all oocytes should be defolliculated now. Shake the tube vigorously
to remove the follicle cells completely, if necessary.
8. Wash the oocytes with Barth’s solution until the supernatant is clear and free of follicular
cell layer fragments.
11.3.5 Selecting Good Oocytes
Rough selection by filtration
1. Fill a 100 ml beaker with 80 ml of Barth's solution. Place the oocyte mesh sieve into the beaker
for grading the oocytes by size. The mesh of the sieve should be completely immersed in the
fluid.
2. Pipette an amount of oocytes onto the sieve. Approximately half the sieve should be covered
with oocytes. Too many oocytes on the filter will lead to an inefficient filtration.
3. Gently move the filter about two centimeters up and down (in the fluid) to separate the
oocytes by size.
4. Transfer the oocytes of appropriate size (that did not go through the sieve) into a 60 mm
petri dish filled with Barth's + gentamicin.
5. The filtered oocytes are incubated at 18 °C for 1 h.
Note: The incubation step is necessary for identifying damaged oocytes in the next step.