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Outgrown the Roost: A Quick Protocol for Passaging Adherent Cells

Your cells have dutifully “sat down”, stuck to the bottom of the dish and replicated, replicated, replicated. You see a swarm of cells that have reached confluency and filled the entire plate. It is time to split them. This can be a relatively easy ritual, but missteps can lead to dead floating cells and the loss of the cell line. A quick protocol is outlined below.

1)     Necessary reagents: a balanced salt solution (BSS), solution to detach cells from the dish (we’ll call this the releasing solution – trypsin and/or EDTA), complete cell growth media, fetal bovine serum (FBS).

2)     Warm up all reagents by placing in a 37°C waterbath for approximately 30 minutes prior to use.

3)     Turn on and set up the tissue culture hood for your use.

4)     Using aseptic technique, remove old spent media from cells and rinse cells with warmed BSS. Depending on how adherent your cells are, you may need to be very careful when rinsing: less adherent cells might detach from the dish if sprayed directly with BSS. In this case, add BSS to the side of the dish carefully and let it flow over the cells.

5)     Remove BSS and add warmed releasing solution. Add a very small volume that is just sufficient to cover the cells as a thin layer. A recommended volume is 1-2mls/25cm2.

6)     Place the plates/dishes, etc. into a 37°C incubator until cells are just detaching from the dish. The amount of time required will need to be determined empirically for each cell line. If cells are particularly resistant to detachment, rinse the cells with the releasing solution once prior to adding more releasing solution and placing in the incubator.

7)     While the cells are in trypsin, I usually set up the plates I am going to move my cells into. Label the required number of plates and place the appropriate amount of warmed growth media into the plates. We’ll discuss seeding concentrations and volumes in another article.

8)     Once cells have released from the bottom of the dish, remove from the incubator and smack (there is really not a better word for this!) the dish with the side of your hand or on the side of any available surface (the incubator works well). Hit hard enough to dislodge all cells from the bottom of the plate, but not hard enough to make the cells bounce up onto the top of the dish/flask. Check to see that all cells have dislodged either by holding the plate up to the light or by looking under the microscope.

9)     In the hood, add a small amount of warmed growth media or BSS+10% FBS to the trypsin and cells, then repeatedly run the liquid in and out of a 5ml pipette to break up any clumps of cells.

10)   Add the cells to a larger volume of growth media or BSS+10% FBS to inactivate the releasing solution and then centrifuge cells.

11)   Resuspend the cell pellet in a small volume of growth media and count the cells using a hemocytometer.

12)   Add enough growth media to bring the cells to the desired density and dispense cells into a prelabeled plate.

13)   Shake/swirl the plate gently to ensure an even distribution of cells.

14)   Place the cells into the 37°C incubator and clean up your hood!

15)   24 hours later check your dishes to make sure your cells have attached to the dish.

16)   Repeat in 2-3 days

If you take the time and passage your cells regularly you will be rewarded with an abundance of cells all waiting for your manipulation!


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