Reagents, supplies, and equipment:

  • M2 medium (Millipore MR-015P-5D)
  • KSOM medium (Millipore MR-020P-5D)
  • 35 mm culture dishes
  • Paper clip for plunger
  • Scissors
  • LN2
  • Long forceps
  • Room temperature water bath

Category
Protocol
Record History
Added on March 7, 2018 at 3:02 PM by Skelton, Jennifer
Modified on March 7, 2018 at 3:25 PM by Skelton, Jennifer
Shared with (contributions)
VCMR: Recovery Protocols Master

Thawing 2-cell stage embryos in straws:

  1. Transfer the straw from the LN2 storage freezer to a smaller container of LN2.
  2. Using forceps, grasp the straw near the label and hold it at room temperature for 40 seconds, then submerge the straw in room temperature water until the ice disappears.
  3. Wipe straw dry.
  4. Holding the straw firmly, cut off the seal and cut through the PVA plug, leaving about half of the cotton plug in place.
  5. Using a metal rod as a plunger, push against the remaining portion of the cotton plug and expel the entire liquid contents of the straw into a 35 mm culture dish.  Do not let the plug drop into the dish.
  6. Wait 5 minutes.  The embryos will shrink considerably.
  7. Transfer the embryos to a drop of M2.  They will rapidly take up water and assume a normal appearance.  Wait 5 minutes.
  8. Wash the embryos in a fresh dish of M2.  Wash embryos through 8 to 10 drops of media.  Place embryos in drops of KSOM overlayed with mineral oil.
  9. Transfer the embryos either into the oviducts of a 0.5 dpc pseudopregnant mouse or culture to the blastocyst stage in KSOM and transfer to the uterus of a 3 dpc recipient.