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This protocol has been written and optimized for picking and deposition of PEG coated polystyrene beads into a 12 well plate. If using other colloids, the coating of the plate and/or the probe needs to be adapted.
- Plasma machine for surface treatment (a Yocto III from Diener electronic is used in this protocol).
- Cytosurge® micropipette with 4µm aperture.
- TC-treated 12 well plates. It is possible to use a non-treated plate. In that case, the plate must be coated with a hydrophilic substance.
- 10µm PEG300 coated polystyrene beads (50mg/ml) (Micromer® 01-54-104 from Micromod®).
- 0.1mg/ml PLL-PEG solution in ddH2O filtrated through 0.2µm pore.
- 0.01M PBS filtrated through 0.2µm pore.
Material and BOT preparation
Place the probe inside the Yocto III apparatus or equivalent:
Apply plasma treatment on the probe for 1 min.
Dispense 1µl of a 1:1 PLL-PEG 0.1 mg/ml solution into the probe reservoir according to Probe preparation instructions.
Probe coating with 0.1 mg/ml PLL-PEG
- Load the probe in the right port of the BOT for gripping.
- In the left port of the BOT, load a plate with one well filled with 1ml of a 0.1mg/ml PLL-PEG solution.
- Perform the Prepare system workflow.
- Use the navigation tool to move the probe to the PLL-PEG containing well. Immerse the probe into the solution and let it stand for 1h.
- Probe is coated and ready to be used for Pick and Place experiment.
As mentioned above, TC-treated plates do not need to be coated.
However, if using untreated plates, PLL coating is required. Add 800µl of a 0.1mg/ml PLL solution per well. After 1h incubation at room temperature, rinse 5 times with PBS.
- Mix 0.5µl of 10µm PEG300 coated polystyrene beads into 1ml PBS (final concentration of 25µg/ml). Dispense this solution into one well.
- Dispense 1ml of PBS in one well. This well will serve to collect the beads after picking
If using the probe directly after coating, the system preparation has already been done. In that case, use the exchange plate workflow to replace the plate containing the coating solution by the one containing the sample. Use the navigation tool to bring the probe in the well that contains the beads. Proceed directly to the Pick and Place step of this protocol.
If using a stored probe, it is necessary to operate a system preparation:
- Place the PLL-PEG coated probe in the probe holder, in the right port of the BOT.
- Place the plate with the sample in the left port of the BOT.
- Perform the Prepare system workflow (without filling the probe and measuring spring constant as it has already been made before storing the probe).
- Use the navigation tool to go over the sample.
Pick and Place
Use the Adhesion workflow to pick up the bead and drop it off, as shown below in the video.
- Select position. Target a bead with the crosshairs. Be sure that the microscope is correctly focused on the bead, otherwise targeting will fail.
- Measure adhesion (grip the bead). Set up the following parameters: Setpoint 20mV, Movement speed 10µm/s, Pause 5s, Pressure -300mbar, retraction distance 50µm.
- Move to new xy position. Use the navigation tool to move the probe to the PBS containing well.
- Approach the surface. Use the Manual z positioning tool to approach the probe to the surface. Recommended approach settings: 20mV for approach setpoint, 10µm/s for approach speed, 100µm/s for retract speed.
- Release the bead. Once the probe is positioned, apply a +500mbar pressure to drop the bead off. It is possible that the bead sticks to the probe. In this case, a new approach to the surface, maintaining +500mbar pressure, will release the bead.
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|Asked: 11/20/18, 7:02 AM|
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|Last updated: 5/14/19, 11:28 AM|