You will need the pre-warmed media, dishes, dispensing pipettor with tips, a pipetting device, a marker pen, timer and cryodevices.
Note that all steps are preformed at room temperature.
Place oocyte(s) in a 20uL drop of MOPS or HEPES buffered holding medium and leave for 1 minute.
Dispense 20uL Equilibration Solution (ES) to the drop and leave for 3 minutes.
Dispense another 20uL ES to the drop and leave for 3 minutes.
Dispense 240uL ES to the drop and leave for 9 minutes.
During equilibration, prepare the cryodevice taking care to maintain sterility. This includes labelling with appropriate witnessing.
Dispense 2x 150uL Vitrification Solution (VS) in to well 2 and 3.
At the end of the equilibration time, transfer the oocyte(s) to the well/dish containing VS1 and leave for 30 seconds.
Transfer the oocyte(s) to the well/dish containing VS2 and leave for another 30 seconds.
At the end of the minute in VS, load the oocyte(s) onto the cryodevice in minimal volume. Remove any excess medium.
Transfer to the liquid nitrogen and stir gently.
Cap the device, taking care to keep the sample under the liquid nitrogen.
