Eppendorf Piezo-actuated Mouse ICSI (intracytoplasmic sperm injection) - Manual (Page 4)

Userguide No 037 | Page 4
Figure 7: Preparation of microinjection capillary. Loosely screw the grip head into the front knurled screw of the actuator (A). Care-
fully push the capillary into the grip head until it touches the stopper (B). Tighten the grip head (C) and rotate the front knurled screw
to align the angled capillary.
5. 1 System optimization prior to ICSI
The TransferMan NK 2 can store up to 3 positions. When a
position is stored, the capillary can be recalled to the posi-
tion automatically simply by pressing the position button
or a double-click on the joystick button. Preinstallation of
positions can significantly speed up the injection process
and reduce the time of cells out of the CO
2
incubator and
eventually improve the survival rate of oocytes.
For mouse ICSI, usually two position storages are used:
Position 1 - Position to perform injection or sperm head
separation
Position 2 – Parking position above the droplets for mov-
ing the plate from sperm droplet to oocytes droplets or vice
versa
Figure 8 : Setting of parameter sets A and B
A B C
4. 2 Preparation of microinjection capillary
If Fluorinert FC-77 or FC-770 is used, back-fill the capillary
using a Microloader. Approx. 1/3 of the capillary is filled with
Fluorinert.
If mercury is used, back-fill the capillary with approx. 2 μL of
mercury (i.e. 4 mm column) using a Hamilton syringe under
a fume hood.
Mount the capillary in the actuator grip head.
Make sure that the capillary goes deep enough and touches
the stopper of the capillary holder.
Absorb a small amount of medium via the front capillary
opening, so that the samples will not get in contact with
Fluorinert or mercury (depending on substance in use),
but the heavy liquid is still as close as possible to the front
opening. This is important to ensure an optimal perfor-
mance for the drilling. Focus on the periphery of the PVP drop, bring the capillaries
into the same focus so that the periphery of the drop and
capillaries are both in focus. Set Position 1 for both capillar-
ies. Optionally, the Z-Limit (special function of the Transfer-
Man NK 2) can be set using the periphery as a guideline to
prevent capillary breakage.
Raise both capillaries, so that they are just above the me-
dium droplet. The capillaries should remain in the mineral oil
to prevent evaporation and capillary blockage. Then Position
2 is set.
The parameters (intensity, speed and number of pulses) of
the PiezoXpert can be preinstalled. Up to 3 sets of optimized
5 Piezo-assisted intracytoplasmic sperm injection (ICSI)