Cell Thawing Protocol


  1. Cryovial containing frozen cells.
  2. RPMI-1640, with 10% FBS, Pen-Strep and L-Glutamine. 9mL/vial to be thawed
  3. 50% RPMI-1640, 50% FBS. 5mL/vial to be thawed
  4. Desired media or buffer


  1. Cells should be thawed immediately when removed from liquid nitrogen or placed on dry ice until ready to thaw.
  2. Bring all thaw reagents to room temperature.
  3. Add 5mL of the 50% FBS/RPMI and optionally 5uL DNAse (10mg/mL) to one 15mL conical tube for every vial being thawed.
  4. Thaw one vial at a time by swirling in a 37°C water bath until only a small ice pellet remains.
  5. Transfer; drop wise, into the prepared 15mL tube.
  6. Add 2-3mL of the 10% FBS media drop wise. If DNAse was not used, add 9mL to better wash off any free DNA. Gently invert tube to mix contents.
  7. Centrifuge at 500xg for 8 minutes to pellet.
  8. Aspirate supernatant. Leave approximately 100uL above the pellet to prevent any accidental aspiration of the pellet.
  9. Resuspend in 1mL desired media and count in Trypan Blue. This will be an approximate count as some cells are in the process of dying. For an accurate count place cells overnight in 37° C incubator and count the next day.


Bloodworks Bio
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Seattle, WA 98104
Phone: 206-568-3637
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