The isolation of cells in suspension and adherent cells (link to the other forum post) requires different mechanical approaches. Suspension cells (e.g., Jurkat), are only held to the substrate by weak non-specific forces – or not at all. When picking up such cells, the force-controlled approach, which works well with adherent samples, often results in the cell slipping away from below the cantilever. To enable the sorting of cells in suspension, we have developed a dedicated workflow that makes fast, easy, and efficient to pick and place cells in suspension.
- FluidFM Micropipette (8 or 4 µm aperture 0.3 N/m stiffness).
- Cell growth media without phenol red (e.g., RPMI-1640 Medium, no phenol red)
Note: For experiments in the OMNIUM system, we recommend phenol red free media to reduce background when imaging.
- In case of no CO2 control within the system use CO2 independent medium (e.g., Leibovitz's L15 medium).
- Complete growth medium (e.g. RPMI-1640 Medium), with 10% FBS and 1% penicillin/streptomycin , filtered (for the newly isolated cells).
- Jurkat cells in suspension.
- Poly-L-lysine (PLL)
- 2x 12-well plates (ThermoFisher-Nunc).
- 1x PBS
Initialization of the system
1. Turn on the microscope components. Turn on the system control unit and the pressure controller.
2. In the computer, open ARYA and create a new experiment.
3. Align the x/y/z axis if necessary.
4. Select the respective plate on the left port (e.g., 12 well plate). Set the content height at 1 mm if the plate used is not listed.
Preparation of the probe
1. Load the probe with 1ul of 1xPBS (filtered).
2. Use the Exchange Plates workflow to move to the right port, place the loading plate inside and place the probe in the loading plate (position A1, A3, C1 or C3).
3. Follow the Preparation or Preparation Advanced workflow until the Go to Sample step.
4. We highly recommend coating the cantilever before the experiment. Follow the protocol “COATING WITH PAcrAm inside FluidFM system” indicated in this link in order to avoid debris and cells to adhere on the surface of the probe.
5. Move to the well containing cells.
6. Maximize Signal and Measure Sensitivity in the Preparation workflow.
Preparation of the cells
1. Shortly before the experiment, remove the medium from the cells and wash them once with PBS. Add then 1 ml of phenol red free medium or CO2 independent growth medium to the cells.
2. Use the Exchange Plates workflow, move to the left port and place this plate in the left port of the FluidFM OMNIUM system.
3. Let the cells deposit on the surface of the well for 10 min.
Preparation of the destination plate
1. Coat a 12 well plate with poly-L-lysine (PLL) according to the manufacturers protocol.
2. Add 1 ml of standard growth medium in each well, where the cells will be placed after the isolation.
3. Once the probe has been loaded into the system, use the Exchange Plates workflow to move to the right port and remove the loading plate.
4. Place the destination plate in the right port of the system.
Preparation of the system
1. Move the probe into the well from which the cells will be isolated.
2. Use the Maximize Signal step of the Preparation workflow. Measure the sensitivity of the probe in an area free of cells.
3. To align the probe, use the Align Probe step in the Preparation workflow and drag the crosshair to the centre of the aperture.
1. Open the Cell Isolation workflow and press on step 1. Pick-Up Cell.
2. Select Aspirate.
3. Select desired parameters. The cell height should be roughly 2 um more than the average cell size in the sample. The default value of 17 um applies for cells with a diameter of 15 um. In parallel, negative pressure of -15mbar will be kept for the duration of the transfer.
4. Go to an area without any cells with the crosshair and press OK. The process begins with a force-controlled approach and will place the cantilever on the preselected height.
5. The settings will appear that allow for so called “fishing” of the cell.
a. Introduce a negative pressure in the Pick Pressure to attract the cell towards the aperture (e.g., -50mbar).
b. Move the cantilever above the target cell.
c. If the cell is not aspired right away, the picking pressure can be adjusted with the Pick Pressure sliding tool.
d. If the cantilever is too high or too low, adjust the step size (start with 1um for safety) and change the height with the arrows.
Note: This is a non-force-controlled movement! Ensure a safe distance from the surface to prevent the cantilever from breaking.
e. Once the cell is in the same focal plane as the cantilever, it is in the center of the tip and is moving steadily with the cantilever, that means it was picked up. To confirm press on Continue.
6. Once the cell has been picked up, select the well in the “destination plate”. The plate is depicted on the right side as it is in the right port. The green label indicates that the well has already been used, but they can also be reused if needed. The blue label depicts a well that will be selected for cell deposition. White wells are empty and free to use. All wells where cells will be deposited need to contain media as mentioned before. Once the well is selected, press Continue.
Note: To confidently achieve monoclonality, an extra washing might be necessary here. You can find out how to wash the extra cells off here.
7. Once the cell is transferred into the destination well check if the cell is still attached to the cantilever. It will appear round as before, in the focal plane of the cantilever. If the cell is still attached the transfer was successful, therefore verify the transfer by pressing OK in the step 2. Verify.
1. In the step 3. Place Cell adjust the parameters with which the cell will be deposited on the bottom of the destination well. The deposition itself is a force-controlled approach. When the setpoint is reached, the cell is kept on the surface for a pre-defined contact time to allow for a mild interaction, while applying the release pressure to break the tight seal with the aperture. Once the contact time is elapsed, the cantilever is retracted first slowly (0.5 um/s) to 20 um, then faster (20 um/s) to 50 um. The cell at this point should have been removed from the cantilever and remained on the bottom of the well. In case the cell is still adhered to the cantilever, the deposition process should be repeated.
Note: The application of a gradually increasing pressure pulse can remove the cell as a final solution. However, viability is seriously compromised upon application of > 200 mbar.
2. After the cell has been stably deposited move away from the cell to the content height using the Navigation Tool.
3. In the step 4. Flush Probe, there is a pressure pulse applied to release the membrane content from the microfluidic channel, that might have been aspirated during the transfer process.
4. After the probe has been flushed move to the step 5. Go to Source. Use the history menu from the Navigation Tool to go back to the initial pool of cells. To pick another cell simply follow again the same workflow from Cell picking and Cell deposition.