Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2014/12/15

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Summary

After starting the SKNSH 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.
After advancing the U2OS cell lines into medium (T-75) flasks, they need to be split into more containers to increase biomass and maintain the cell line. If kept in the same container for too long, cells will enter senescence and no longer be competent for transfection. Therefore it's important to continue splitting cells and to experiment on cell lines that are still relatively young.
It's important to keep in mind that not all cell lines develop at the same rate. The U2OS line grows significantly faster than SKNSH, and will therefore require more frequent splitting/passaging.

Goals

  • Passage U2OS cells from PS-β to PS-1
  • Advance SKNSH cells from PS-α to PS-β

Protocol

advancing adherent cells
passaging adherent cells

Materials

  • Active, adherent U2OS cells in T-75 flask
    • high confluence is ideal (cells have grown to form a complete monolayer on the flask with few gaps)
  • Active, adherent SKNSH cells in T-25 flask
    • high confluence is ideal (cells have grown to form a complete monolayer on the flask with few gaps)
  • Complete medium for U2OS (McCoy's 5A w/ 10% FBS & 1% pen/strep)
  • Complete medium for SKNSH (Eagle's Modified Essential Medium w/ 10% FBS & 1% pen/strep)
  • 1x phosphate-buffered saline (PBS)
  • trypsin-EDTA
  • T75 flasks
  • pipette tips
  • 70% ethanol in spray bottle

Notes

Observations, measurements, placeholder info etc. go here

Results/Conclusions

Any significant data goes here. Thoughts on what happened, significance of findings, what to do next etc.
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