Freezing Adherent Cell Lines
Carolina Lopez
Published: 2024-03-21 DOI: 10.17504/protocols.io.5qpvokxq9l4o/v1
Disclaimer
Abstract
This protocol describes how to freeze Adherent cells. Examples of these cells are: A549 cells, LLCMK2 cells, and MDCK cells.
Steps
Freezing Adherent cells
1.
- Wash flask 2x with sterile PBS.
- Add 2mL of trypsin/T75. Incubate at 37 oC for 2-3 mins or until cells are detached from the flask.
- Add 8mL of cell culture media and pipette up and down. Transfer all the media to a 15mL tube.
- Centrifuge at 1200 rpm for 5 min.
- Discard supernatant and resuspend cell pellet in 1mL of cell culture media. Add 9mL of media and pipette up and down to homogenize.
- Count the cells. You will need your cell concentration/# for step 6.
- Spin the tube containing the cells in medium again at 1200 rpm for 5 min.
- Meanwhile, prepare the freezing medium. The amount of freezing medium to prepare should be the amount needed to dilute the cell pellet so that you have a final concentration of 1*106 cells/mL.
- 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.
- 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)
- Transfer the cells to the Stratagene chamber at 4C and then transfer the chamber to the -80C for 1 day. After 1 day, transfer the tubes to the liquid nitrogen. You can keep some vials in the -80C. These could last for at least 2-3 years.
