WinterVomitingLab:Protocols/Passaging HeLa cells

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Passaging HeLa Cells.


  • HeLa (Henrietta Lacks) cells (ATCC # CCL-2)
  • Complete MEM
    • MEM (Fisher- cat# SH30024LS)
    • 10% Atlanta Bio Fetal Bovine Serum (Atlanta Biologicals- cat# S11050H)
    • 1% Penicillin/Streptomycin (VWR- cat# 12001-692)
    • 1% Sodium Pyruvate (Fisher- cat# SH3023901)
    • 1% Non-essential amino acids (VWR- cat# 12001-634)
  • PBS
  • Trypsin


  1. Determine the number of new flasks to be made, and label with passage #, cell line, date, and initials. Add the appropriate amount of fresh media for the size flask you are using (T25- 7ml, T75- 25ml, T175- 50ml), minus the amount you will be adding from the passaged flask. HeLa cells are passaged at a ratio of 1:10.
  2. Pour media from each flask into the waste beaker, cover beaker, and set aside.
  3. Rinse flasks with PBS, collecting the rinsate in the waste beaker.
  4. Add 3 ml trypsin (T175 flasks) and rock flask to cover the bottom with a thin layer of trypsin.
  5. Place flasks in 37°C incubator, checking every 2 minutes, until all cells have detached from the bottom. If the cells do not detach easily, gentle agitation may be necessary.
  6. Add 7 ml of fresh media and thoroughly rinse the flask to remove and mix all cells, then aliquot them into the appropriate number of new flasks containing fresh media.
  7. Discard old flasks and place new flasks in the 37° C incubator.


Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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