Aug 23, 2024

Splitting Adherent Cell Lines V.2

DOI
https://dx.doi.org/10.17504/protocols.io.x54v9p51qg3e/v2
  • 1Washington University
Carolina Lopez
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DOI: https://dx.doi.org/10.17504/protocols.io.x54v9p51qg3e/v2
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: August 22, 2024
Last Modified: August 23, 2024
Protocol  Integer ID: 106114
Keywords: splitting adherent cell line, adherent cell lines this protocol, adherent cell lines in culture, mdck cell, adherent cell line, llcmk2 cell, a549 cell, cell, examples of these cell, line
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
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%

Protocol
Splitting Adherent cells
  1. Wash flask 2x with sterile 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 media and resuspend cells in 10mL Tissue Culture Medium
  6. 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.