Wash the cells with sterile PBS (w/o Ca++/Mg++) or HepesBSS
Add undiluted accutase to the culture vessel (2 ml per 25 cm2)
Incubate at room temperature for 5-15 min or at 37°C for faster detachment
When the majority of the cells has detached, centrifuge the suspension and resuspend the pellet in fresh medium. In most cases, no additional washes or neutralization steps are required.
