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Handling EmbryoMax Mouse Embryo Media



Handling EmbryoMax Frozen Mouse Embryo Media

EmbryoMax Frozen Mouse Embryo Media are formulated for optimal, consistent performance. These media are complete, pH adjusted, and ready to use after thawing. Media are shipped and stored frozen to extend their shelf life.
Protocol
Prepare a Bottle of Frozen Mouse Embryo Medium for Use
  1. Frozen media can be placed in a refrigerator (2°C to 8°C) to thaw overnight. The thawed media should be swirled gently to mix the media, and warmed to 37°C prior to use. Alternatively, place frozen medium into a 37°C water bath until thawed completely. It is best to gently swirl the medium during thawing to accelerate the process, and mix the medium. Do not allow the thawed medium to remain in the 37°C water bath for extended periods after it has thawed completely, and warmed to 37°C.
  2. Once thawed the medium has a shelf life of 2 weeks, and must be stored at 2°C to 8°C between uses. Do not re-freeze the medium. Medium stored beyond this 2 week period may not perform optimally.

Note: The “Last Thaw Date” on the label indicates the last day in the shelf life of the frozen media. Media stored frozen beyond this date may not perform optimally. Media are best used prior to the “Last Thaw Date;” however, once the “Last Thaw Date” has been reached, the media should be thawed, stored at 2°C to 8°C, and used within 2 weeks.

Note: Although the pH of these media are correct when thawed, the pH of the sodium bicarbonate buffered formulations will rise as the media are exposed to air. This rise in pH will increase with the frequency of opening the bottle, and is particularly rapid when drops of medium placed on a dish for embryo culture are exposed to air. Thus, it is recommended that the drops of medium (placed under light mineral oil) are pre-equilibrated 6 hours to overnight in a humidified incubator at 37°C, in a 5% C02 + 95% air atmosphere, prior to introducing embryos into the medium.


Handling EmbryoMax Powdered Mouse Embryo Media


Protocol
Powdered Medium 50 mL Size
  1. Medium is packaged as five bottles of powdered medium and five bottles of sterile diluent per box. Remove one bottle of powder and one bottle of diluent. Check that the catalog number on both the diluent and powder are the same.
  2. Using a sterile pipette, withdraw 50 mL of liquid from the bottle of diluent. These bottles have been filled with 52 mL of diluent to allow accurate withdrawal of liquid.

    Note: Note: It is important to withdraw an accurate volume of diluent and to add the 50 mL of diluent accurately to the powder. Improper volume of diluent will affect the pH and osmolarity of the hydrated medium.

  3. Gently rock the vials end-to-end to hydrate the powder. DO NOT SHAKE. The medium contains BSA and will foam if shaken. The BSA in the medium may take up to 15 minutes to completely dissolve at room temperature. After the initial mixing, occasionally swirl the medium until it is completely in solution. Getting the powder completely into solution can be accelerated by placing the vial of hydrated medium in a 37°C water bath and occasionally swirling the medium until it is completely in solution.
  4. Draw the medium into a 50 cc syringe and filter through a 0.2 micron syringe filter.
  5. The medium is now ready for use.
Recommendations for Use
  1. Media that contain NaHCO3 will rapidly become alkaline upon exposure to air. Drops of medium under sterile light mineral oil should be equilibrated overnight in a 37°C incubator in a 95% air + 5% CO2 humidified atmosphere. The transfer of gas to the media drops under light mineral oil occurs slowly.
  2. The 5% CO2 concentration in the incubator is important. Routinely calibrate the CO2 in the incubator with a Fyrite unit or similar calibration instrument.
  3. Media that contain HEPES are formulated for use in air, and can be used directly out of the vial without any pre-incubation.
  4. We recommend that you use a fresh preparation of medium for each experiment. If you need to store media for future use, store the hydrated medium at +4°C in the dark for up to one week.

References

  1. Quinn, P., Barros, C., and Whittingham, D.G., Preservation of hamster oocytes to assay the fertilization capacity of human spermatozoa. J. Reprod. Fertil. 1982; 66:161-168.
  2. Lawitts, J.A. and Biggers, J.D., Methods in Enzymology 1993; 225.

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