Eppendorf Piezo-actuated Mouse ICSI (intracytoplasmic sperm injection) - Manual (Page 6)

Userguide No 037 | Page 6
Fig. 10: Mouse ICSI
Move the stage to the injection droplet. Place the holding
capillary using the previous stored positions. The oolemma
of the oocyte should be sharply focused at 20x or 40x.
Aspirate and hold the oocyte using the holding capillary
and place the polar body at the 6 or 12 o’clock position.
Use Position 1 to recall the injection capillary and bring the
injection capillary to the zona pellucida (Figure 10 A);
(Y-OFF function can be activated on TransferMan NK 2
control board to reduce the lateral movement of the capil-
lary while penetrating the cell which may cause cell lysis).
Advance the injection capillary while applying piezo impulse
parameter set A via the foot control to penetrate the zona
(Figure 10 B). Try to expel the zona plug into the perivitelline
space (Figure 10 C). Subsequently, push the oolemma till a
funnel shape is seen. Move one sperm head forward using
A B
C
D E F
G
the dispensing function of the CellTram vario until it is close
to the tip of the capillary and advance the capillary until it
almost reaches the opposite side (Figure 10 D). Then trigger
piezo drilling parameter set B via the foot control until the
relaxation of the oolemma is observed (Figure 10 E). The
sperm head is then injected with a minimum of medium
(Figure 10 F). Withdraw the capillary gently (Figure 10 G).
Release the injected oocyte. Repeat the procedure for all
other oocytes.
5.3 Post-ICSI
After injection, transfer the oocytes in KSOM under mineral
oil in a humidified 5 % CO
2
incubator at 37 °C and further
treat as described elsewhere [4].