Accutase is formulated at a concentration that is ready to use, once defrosted. (Note: Never defrost a bottle of Accutase at 37 °C.) A defrosted bottle of Accutase can be removed from the refrigerator and immediately applied to cells. It does not need to be and should not be pre-warmed to 37 °C. Accutase contains proteolytic and collagenolytic enzymes to gently break down the cell adhesion structure on the outside of cells that attaches them to the bottom of the flask.
Note: Don't try and be thrifty and kill your cells. Enough Accutase must be added to cover the bottom of the flask. Do not add a small amount of Accutase and then pipette it up and down to remove the cells. Pipetting up and down kills cells!
This entire procedure should be done in a laminar flow hood using proper aseptic technique.
- Carefully aspirate all of the media from the cell culture flask. (Rinsing with PBS is not necessary.)
- Immediately add enough Accutase to the flask to cover the cells. (Typically 2.5 to 5ml for a T25 flask depending upon confluency and density of the cell culture.)
- Set the flask aside at room temperature (RT) for 5 to 10 minutes up to a maximum of 1 hr. Check the flask frequently to see if the cells have rounded rather than merely shrunken or no longer appear “spidery” while remaining attached to the bottom of the flask.
- Once the cells have turned into “balls”, smack the flask against the palm of your hand to dislodge any “stickers”.
- Gently disperse the cells and take a sample of the cell suspension to determine the viable cell density.
- Add an aliquot of the detached cells to fresh media in new flasks. Place the flasks into the 37 °C incubator. No neutralization steps are required. The cells will reattach within a few minutes depending upon cell type.