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Cell passage: How to correctly dilute and split cultured cells

There are many different cell culture techniques. Utilizing correct cell passaging methods is important to keep cell line in exponential growth curve, therefore making it a good model as a host for transfection experiments. “Cell passage” is a term used by other scientists to demonstrate the following process:

  1. Wash cells with PBS
  2. Detach cells from flask by trypsinization
  3. Resuspend in complete media (contains FBS) to neutralize trypsin
  4. Transfer appropriate dilution to new flask containing ample media

The correct passage dilution is cell line specific and depends on factors such as doubling time and intended use of the cells.  The important aspect to remember is that the split ratio is determined from the total volume of trypsin and media from steps 2 and 3 above.  As an example for a T75 flask, if 1 mL of trypsin is used to detach the cells and 9 mLs of complete media is used to neutralize the trypsin, then the total suspension volume is 10 mL.  Depending on the cell line, here are examples of split ratios a researcher might follow:

  • 1:10 split: transfer 1 mL of the suspension to a new T75 flask and add complete media
  • 1:5 split: transfer 2 mL of the suspension to a new T75 flask and add complete media
  • 1:2 split: transfer 5 mL of the suspension to a new T75 flask and add complete media

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