Depending on whether or not temperature control is important, two solutions have been implemented for perfusion and solution exchange:
When temperature control is not required, solutions may be exchanged via a 4-channel manifold ( ALA scientific) mounted on a pipette holder in a coarse manipulator (Narishige). The 4-channel manifold is connected to a custom-build valve bank fed by a peristaltic Gilson pump.
If temperature control is important, the facility has acquired 6-channel heated manifolds from Havard Scientific. These devices are run from the same valve banks and peristaltic pump but offer very accurate temperature control of the solution delivered, with the option to regulate from an external temperature probe (to position inside the bath for example). We plan to have the valve controllers interfaced with the microscope acquisition software for seamless synchronization with the imaging.
In every case, excess solution is removed via a small aspirator connected to a trap and th building vacuum. The trap must be cleaned after every experiment! For long-term experiment, we recommend using two sets of input and output. The tip of the manifold and one suction line positioned as close as possible to the front lens of the objective, a simple perfusion line with fresh media and a second vacuum line positioned at the edges of the dish. This configuration ensures both localized drug exposure and maximum viability of live cells.
