Multichannel Systems HiClamp Manual - Manual (Page 72)

HiClamp Manual
66
9.3 Plating Oocytes
You need a well plate filled with Barth's + gentamicin ????
Important: Visually check all well plates before use. Use only unsterile and very flat plates.
Sterilization may lead to warped plates. Warped plates will decrease the injection and expression
efficiency.
1. Aspirate a number of oocytes from the dish with a transfer pipette by applying a low negative
pressure, for example, with a rubber ball.
2. Move from well to well with the transfer pipette, carefully dropping one oocyte into each of the
wells of the well plate. Grip only the glass pipette and do not squeeze the rubber ball, to avoid
that too many oocytes will leave the pipette. The oocyte should drop into the fluid by its weight
alone. If occasionally more than one cell drops into a well, this can be corrected after the plating
will have been finished.
3. Due to the higher density of the vegetal pole, the oocytes will usually settle with the animal pole
facing up if the (sticky) follicle cells were completely removed. Immediately check the position of
each oocyte under a stereo microscope, and correct it carefully with the golf-club shaped glass
tool, if the orientation is not correct. A manual correction should be necessary for less than five
percent of the oocytes if the follicle cells were completely removed.
4. Cover the well plate with its lid, and seal the rim with Parafilm to reduce evaporation, and store
it at 18 °C until injection. It is possible to inject the cells right after plating, but the oocytes should
not be further disturbed, for example not be washed or otherwise treated until they have been
allowed to adhere to the well bottoms (about 2 to 3 hours). The oocytes can also be injected on
the next day, after being stored overnight at 18 °C. If injected on the next day, it is recommended
to check the viability again under the microscope, and replace bad oocytes before the injection.