Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2014/12/12

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After starting the cell lines in small (T25) flasks, they need to be transferred to a larger container to increase biomass in preparation of experimentation. If the initial thawing and starting of a cell line is Passage Alpha, the first transfer into a T75 flask is Passage Beta. The cells will need to have their medium removed, their attachment to the flask disrupted with trypsin-EDTA, and then be transferred into fresh media.


  • Advance U2OS cell line into T75 flask


advancing adherent mammalian cells


  • Active, adherent U2OS cells in T25 flask
    • high confluence is ideal (cells have grown to form a complete monolayer on the flask with few gaps)
  • Complete medium (McCoy's 5A w/ 10% FBS & 1% pen/strep)
  • 1x phosphate-buffered saline (PBS)
  • trypsin-EDTA
  • T75 flask
  • pipette tips
  • 70% ethanol in spray bottle


Addition of PBS is to wash the cells of any residual medium, which can interfere with the trypsin-EDTA digestion.

Trypsin-EDTA breaks the cell's adhesion to the flask bottom. An easy visual indicator is when the frosted appearance of the flask bottom starts to break up and move around. Washing the flask bottom with medium prior to transferring to the new vessel also helps with this process.

Do not leave the cells in the trypsin-EDTA solution for too long or the cell membranes will rupture. Adding the fresh medium inhibits the trypsin-EDTA digestion.


U2OS culture is labeled PS beta and is in the upper incubator, middle shelf.