1. Set up thawing dishes (as shown in diagram):
- At 37°C: Aseptically dispense a minimum volume of 250 μL of TS and warm to 37°C (incubator without CO2) or on a heating stage at least 30 minutes prior to starting warming procedure.
Note: For oocytes, dispense a minimum of 1 mL of TS
2. Identify HSV Straw(s) to be warmed from LN2 storage and quickly transfer to an LN2 filled holding reservoir in preparation for warming procedure.
3. Place LN2 filled holding reservoir in close proximity to the working area and stage of the microscope in order to achieve subsequent rapid manipulation from reservoir to TS.
4. Remove TS dish from 37°C incubator or heating stage and place it under focus on top of the microscope stage.
5. Lift the straw with forceps enough to expose the colored handling rod. Make sure the end with the specimen(s) remains immersed in the LN2.
6. Use a Knipex (or other wire cutter device) to cut the straw at the height of the colored handling rod. The red cut-length guide on the Knipex should be positioned in maximum length position or removed.
- Alternatively, use fingers and thumb to spin the straw while making cutting movements with scissors, 10 mm under the top of the colored handling rod.
7. With one swift but controlled motion, quickly grab the handling rod and extract it out of the straw.
8. Immediately plunge the curved spatula (or gutter) of the handling rod into the 37°C TS and gently swirl to detach specimens from device and leave the oocyte or embryo for a total of 1 minute in the TS. After 30 seconds following the initial plunge, gently pipette the specimen (if floating) and place at the bottom of the TS drop/well.
Steps 9-12 must be performed at room temperature (22-27°C).
- At room temperature: Aseptically dispense one (1) 50 μL drops of DS on a sterile Petri dish
9. Transfer specimen(s) to DS for 4 minutes. Gently pipette specimens once to ensure complete rinse in DS.
10. During the 4 minute exposure in DS, aseptically dispense two (2) 50 μL drops of WS (WS1, WS2) as shown in diagram.
11. Transfer specimen(s) to each WS1 then WS2 for 4 minutes each, undisturbed.
12. Transfer warmed OOCYTE(S) to pre-equilibrated culture medium with 20% (v/v) protein supplement or 12 mg/mL for recovery (2-3 hours to allow for spindle reformation) prior to subsequent manipulations.
13. There are two options for warmed EMBRYO(S):
a) For immediate transfer to patient: transfer EMBRYO(S) to pre-equilibrated ‘transfer’ medium containing 20% (v/v) protein supplement or 12 mg/mL.
b) For further culture: transfer EMBRYO(S) to pre-equilibrated culture medium containing 20% (v/v) protein supplement or 12 mg/mL for a 3 hour recovery period. After recovery period transfer EMBRYO(S) to culture medium with 10% (v/v) protein and incubate accordingly until desired developmental stage has been reached for transfer to patient.