Multichannel Systems HiClamp Manual - Manual (Page 71)

Service and Maintenance
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9.2 Preparation of 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.
Fine selection
Use a stereo microscope and the golf-club shaped glass tool to check each single oocyte
for the following criteria.
Outer form:
 No visible damage of the cell.
 Well separated colors (dark and light brown).
 No residues of follicular tissue.
Size:
Uniform size of about 1.2 mm.
Note: Selecting oocytes is an important step. Perform it very carefully to obtain best results.