In this protocol, the FluidFM probe is coated with PAcrAm (SuSoS AG). This compound is a polymer used as antifouling agent for glass and polymer cell culture substrates. This coating is ideal for coating micropipettes in isolation workflows, but it also can be used for Nanosyringe coating for the injection of specific compounds. PAcrAm is distributed in 0.1 mg lyophilized aliquots, which also contain the buffer required for surface modification. Aliquots can be purchased directly from Cytosurge.
There are two different protocols for probe coating using PAcrAm. Using protocol a), the inside of the microchannel is also coated (recommended for injection and extraction), while using b), PAcrAm only absorbs on the exterior surface of the cantilever (recommended for pick and place). The a) method can only be applied to one cantilever at a time.
a. COATING WITH PAcrAm inside FluidFM system1. Temperate the PAcrAm aliquot to 37 degrees. Dilute the content in 1 ml of 37 degrees ultra-pure water. Vortex for 5 s.
2. In a 12-well plate (ThermoFisher, Nunc), dispense all the PAcrAm solution into the position A1.
4. Fill the reservoir of the FluidFM probe with 1 ul of your solution (e.g. for isolation workflow, use Trypsin 10x).
5. Place the probe in the probe loading plate.
6. Select Preparation or Preparation advanced workflow and follow it until the step Go to Sample. In this step, right after filling the probe, dip the FluidFM probe into the PAcrAm solution on the plate placed in the left port (A1 position), using the Navigation Tool.
7. Apply -250 mbar pressure for 5-10 seconds. This step makes sure that the inside of the channel is also coated.
8. Incubate the probe in PAcrAm solution for 30 min while applying -20 mbar in the cantilever.
9. Use the washing tool to wash the probe in 2 wells of ultra-pure water and three wells of PBS. The probe is now ready to use.
b. Pre-COATING Probes WITH PAcrAm
1. Temperate the PAcrAm aliquots to 37 degrees. Dilute the content of each aliquot in 1 ml of 37 degrees ultra-pure water. Vortex for 5 s.
3. Place the FluidFM storage plate on top of the 12-well plate.
4. Place the probes to be coated into the storage plate above the Param containing wells in a way that the bottom is immersed into the coating solution.
5. Incubate for 30 min at room-temperature.
6. Wash 3 times in ultra-pure water by dipping the probe for 5 sec in the other 3 wells (A2, A3, A4).