COS-7 Cell Culture Protocol

The COS-7 cell line grows adherently to glass and plastic in cell culture conditions.  COS-7 cells are typically grown in a complete growth medium such as Dulbecco’s MEM (i.e. DMEM) and modified with the addition of 10% FBS.  Cells should be incubated at a temperature of 37°C in 5% CO2.  COS-7 cells should be subcultured according to the protocol below with a subcultivation ratio of 1:4 to 1:8.  Culture medium should be renewed 2-3 times per week.

Subculture Protocol for COS-7

  1. Rinse cells with sterile 1x PBS to remove all traces of media containing FBS
  2. For a T75 flask, add 2 mL of Trypsin-EDTA and watch for cell layer detachment using an inverted microscope.  Detachment of cells should occur within 3-15 minutes.  If cells are not detaching, put the flask in the incubator to increase enzymatic activity.  Do not agitate cells during cell detachment procedure since agitation encourages clustering.
  3. After detachment, add 8 mL of complete medium to neutralize the trypsin.
  4. Add the appropriate split ratio aliquot of the cell suspension to a new culture flask.
  5. Incubate at 37°C in 5% CO2. Maintain culture at a confluency between 30-90%.

COS-7 cells doubling time is 35-48 hours.  For long-term storage, COS-7 cells should be stored in the complete growth medium (DMEM with 10% FBS) modified with 5% (v/v) DMSO and stored frozen in the vapor phase of liquid nitrogen.


  • Passage Number: Cells undergo genotypic variations due to high passage number in culture.  Always record passage numbers on the flask and do not allow high passage numbers. Cells will become slow growing and hard to transfect, along with data sets that cannot be repeated as a result of the changing genetics of the cell.
  • Confluency: Cells in flasks must be grown within certain ranges of confluency.  Too few cells result in lack of cell growth due to lack of cell contact; too many cells in the flask will cause the cells to stop proliferating from a lack of available room to grow in flask.  Cells should maintained at exponential growth by keeping confluency between 30-90%.
  • Cells not detaching from flask: If cells do not detach from flask during trypsinization step, one of the following may be to blame: 1) dissociation agent is too weak; try a higher concentration and incubated the flask at 37°C to increase enzymatic activity, 2) cells are too confluent creating tight junctions; maintain cultures at <90% confluency, and 3) complete medium was not washed from flask prior to addition of trypsin; ensure complete washing of flask or perform consecutive PBS washes.


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