Aug 23, 2024

Public workspaceFreezing Adherent Cell Lines V.2

DOI
dx.doi.org/10.17504/protocols.io.5qpvokxq9l4o/v2
  • 1Washington University
Carolina Lopez
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DOI: dx.doi.org/10.17504/protocols.io.5qpvokxq9l4o/v2
Protocol Citation: Carolina Lopez 2024. Freezing Adherent Cell Lines. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvokxq9l4o/v2Version created by Sydney Faber
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 21, 2024
Last Modified: August 23, 2024
Protocol Integer ID: 106113
Abstract
This protocol describes how to freeze Adherent cells. Examples of these cells are: A549 cells, LLCMK2 cells, and MDCK cells.
Materials
FREEZING MEDIUM:
90% of TCM
10% of DMSO

TISSUE CULTURE MEDIUM (TCM): Filter through 0.2 µm filter
ComponentAmountConc. Supp.Product information
DMEM500 mLGibco Cat. # 11965092 (#11965118-cs)
Gentamicin500 µL 50µg/mLGibco Cat #15750060 (#15750078-pk) – 50 mg/mL
Sodium Pyruvate 5.0 mL1mMCorning Cat #25-000-Cl – 100mM
L-Glutamine 5.5 mL 2 mMSigma Aldrich Cat #G7513 – 200mM
FBS50 mL10%

DMSO (Dimethyl sulfoxide): Sigma-Aldrich Cat# D2650
Freezing Adherent cells
Freezing Adherent cells

*Try to freeze cells at the lowest passage possible and confirm the cells are negative for mycoplasma before freezing.

  1. Wash flask 2x with sterile 1X PBS.
  2. Add 2mL of trypsin/T75. Incubate at 37ºC for 2-3 mins or until cells are detached from the flask.
  3. Add 8mL of cell culture media and pipette up and down. Transfer all the media to a 15mL tube. 
  4. Centrifuge at 1200 rpm for 5 min. 
  5. Discard supernatant and resuspend cell pellet in 1mL of cell culture media. Add 9mL of media and pipette up and down to homogenize. 
  6. Count the cells. You will need your cell concentration/# for step 6.
  7. Spin the tube containing the cells in media again at 1200 rpm for 5 min. 
  8. Meanwhile, prepare the freezing medium (see materials for recipe). Prepare the amount of freezing medium needed to dilute the cell pellet so that you have a final concentration of 1*106 cells/mL. 
  9. Discard the supernatant and resuspend the pellet in 1mL of freezing medium. Add the remaining volume of the freezing medium. Make sure you homogenize the solution well. 
  10. Add 1mL of freezing medium containing 1*106 cells/mL to cryotubes (caps screw from the outside). Label all the tubes with the following information:
  • Cell Name
  • Generation/Passage
  • Date
  • Name
  • Cell concentration (if there is space)
11. Transfer the cells to a Mr. Frosty Freezing container containing isopropanol and then transfer the container to the -80ºC for 1 day. After 1 day, transfer the tubes to the liquid nitrogen. You can keep some vials in the -80ºC for a couple of months, however, the -80ºC is not for long-term storage.