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Thaw Embryos in Vials

Reagent and supplies

ReagentSourceCatalog Number
EmbryoMax M2 mediumSigmaMR-015-D
EmbryoMax KSOM AdvancedSigmaMR-101-D
EmbryoMax mineral oilSigmaES-005-C
35 mm tissue culture dishesFisher0877230
60 mm tissue culture dishesFisher0877231
PBSLonza17-512F
ScissorsFisehr08950
P-200 pipetmanRaininP200
Wide bore pipette tipsVWR76635-646

Prepare culture dishes (one set per straw thawed)

  1. Set aside one 35 mm dish for depositing thawed embryos in.
  2. Prepare one 60 mm dish containing 8-10 drops of 50 μl M2 medium for embryo washing.
  3. Prepare one dish with KSOM drops overlaid with mineral oil for embryo culture.

Thawing 8-cell stage embryos in vials (frozen using JAX slow freeze method):

  1. Remove vial from LN2 storage and thaw at room temperature for 12 to 15 minutes.
  2. When completely thawed, add 0.8 ml of PBS dropwise to dilute the DMSO.
  3. Withdraw contents using a P200 Pipetman and wide-bore pipette tip and place in 35 mm dish.
  4. Recover embryos, count, and place in a fresh drop of M2. Wash embryos through 8 to 10 drops of media.
  5. Place embryos in drops of KSOM overlaid with mineral oil.
  6. Embryos may be cryopreserved, cultured, or transferred to 0.5 dpc recipients.