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Thawing Cells - Slow

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol describes how to thaw cells that were frozen down in the liquid nitrogen or -80C Freezer. After thawing, cells should be ready to be cultured or used in an experiment. This is the Slow Variant of this protocol.

Reagents and Materials

  • Thaw Media
  • Frozen down Cell Aliquots
  • 50mL Centrifuge Tube

Things to Note before starting

Estimated Timing

This protocol takes ~20-40 minutes as it has multiple room temperature incubation steps. 1

Procedure

  1. Remove Cell Aliquots from their long term storage in the -80C Freezer or the Liquid Nitrogen Tank.
  2. Using the floating foam tube rack, float the Cell Aliquot in the Hot Water Bath. 2
  3. Add 1mL of Thaw Media into a sterile 50mL Centrifuge Tube.
  4. Monitor the Cell Aliquot every 3 minutes. When the Aliquot has only a tiny crystal of ice remaining at the bottom, remove it from the hot water bath. 3
  5. Using a p1000, transfer the Cell Aliquot into the 50mL Centrifuge Tube containing 1mL of Thaw Media. Place that tube on ice. This tube shall be henceforth referred to as the "Thaw Tube".
  6. Mix by swirling the Thaw Tube.
  7. Incubate on Ice for 3 minutes.
  8. Using a p1000, wash the Cell Aliquot tube with 2mL of Thaw Media, and transfer the washed media into the Thaw Tube.
  9. Mix by swirling the Thaw Tube. Incubate on Ice for 3 minutes.
  10. Add 4mL of Thaw Media into to the Thaw Tube. Mix by swirling the Thaw Tube. Incubate on Ice for 3 minutes
  11. Add 8mL of Thaw Media into to the Thaw Tube. Mix by swirling the Thaw Tube. Incubate on Ice for 3 minutes
  12. Add the rest of the Thaw Media into to the Thaw Tube. Mix by swirling the Thaw Tube. Incubate on Ice for 3 minutes.
  13. Spin down the Thaw Tube at 800 x g for 5 minutes. A Pellet should be visible at the bottom. Aspirate to the pellet.
  14. Resuspend the cells in appropriate media for culture or experiment.

Next Steps

The viability of the cells should be assessed by counting the cells with the Countess. After the recovery of the cells has been assed, the cells can be placed in the appropriate culture or used directly within an experiment.

Appendix

Recipe's for Buffers

Thaw Media

This buffer is used to recover cells from liquid nitrogen storage. The recipe below is buffer to thaw 1 sample. Make each aliquot of this media in it's own 50mL Centrifuge Tube. This buffer should be kept on ice while being used.

  • 31.5mL of Heat Inactivated FBS
  • 3.5mL of DNase I

Footnotes

  1. It would be smart to ask what is the downside to a fast protocol. There has been anecdotal evidence that the fast nature of this protocol causes some shock to the cells. The immediate exposure to the large volume of chilled media might be toxic to some cells - causing reduced viability. No significant viability loss has been demonstrated at the time of writing this protocol. However, if you are working with previous cells and need the best recovery chances, I would recommend using the slower variant. Speak with CB Wolf or anyone in the Mouse Core for the up to date recommendations. ↩

  2. The floating foam tube racks can be located in the top drawer where the hot water bath is located. If you are having difficulty finding it, speak to someone in the Mouse Core (Heather, CB, or Mitch at the time of writing this protocol) ↩

  3. The goal of this step is to not over thaw the Cell Aliquot using the hot water bath. Taking the Cell Aliquot out when there is only a tiny sliver of ice allows us to be confident that the Cell Aliquot was not sitting in the Hot Water Bath for longer than necessary. ↩