Aug 22, 2024
Version 2
Splitting Adherent Cell Lines V.2
- 1Washington University
Protocol Citation: Carolina Lopez 2024. Splitting Adherent Cell Lines. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9p51qg3e/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: December 17, 2023
Last Modified: August 22, 2024
Protocol Integer ID: 106114
Disclaimer
Timing for trypsin treatment and following splits may need to be adjusted based on the different cell types considering how adhesive they are and their growth rate.
Abstract
This protocol describes how to split and maintain adherent cell lines in culture. Examples of these cells are: A549 cells, LLCMK2 cells, and MDCK cells.
Materials
TISSUE CULTURE MEDIUM (TCM): Filter through 0.2 µm filter
| Component | Amount | Conc. Supp. | Product information | |
| DMEM | 500 mL | Gibco Cat. # 11965092 (#11965118-cs) | ||
| Gentamicin | 500 µL | 50µg/mL | Gibco Cat #15750060 (#15750078-pk) – 50 mg/mL | |
| Sodium Pyruvate | 5.0 mL | 1mM | Corning Cat #25-000-Cl – 100mM | |
| L-Glutamine | 5.5 mL | 2 mM | Sigma Aldrich Cat #G7513 – 200mM | |
| FBS | 50 mL | 10% |
Protocol
Protocol
Splitting Adherent cells
- Wash flask 2x with sterile PBS.
- Add 2mL of trypsin/T75. Incubate at 37ºC 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 media and resuspend cells in 10mL Tissue Culture Medium
- Add 2mL of cell suspension to T75 flask, then add 8mL Tissue Culture Medium. The cell will be ready for next split two days later.