Ways To Ensure Experimental Consistency #1

When reproducing experiments, it is important to keep experimental variation and human error to a minimum.

Cell-line Stocks

One way to ensure consistency is to keep track of the passage numbers of your cell-lines. When you order in new cells or are given cells from a collaborator, make a note of how many passages they have already been through.  Once this is done, proceed to create your own stocks. I have typically used the organization as set out below.

The Master cells are cells that you expand upon receipt. Freeze a high concentration (e.g. ~1x10⁷ cells/ml) of these down as your Master cell stocks and save an aliquot to continue growing for the Submaster cell stocks. For the Submaster stocks, you can freeze at a lower cell concentration (e.g. ~5x10⁶ cells/ml). After freezing down aliquots of your Submaster cell stocks, once again, save an aliquot for further expansion for your Working cell stocks. The Working cells can be frozen anywhere between 1-5x10⁶ cells/ml.

It is intended that the Working cells be used for x number of experiments before a new vial is thawed. It is only once you exhaust the Working stocks that you thaw out one vial of your Submaster cell stocks for expansion to create a new Working cell stock. From this you can see that it is only once your Submaster stocks are exhausted do you then thaw out one of your Master cells vials to repeat the entire process.

Keep a log book of your cell stocks and make sure that the numbers are regularly updated.