FluidFM micropipettes can be used to separate cells with specific properties or to avoid serial dilution procedures in isolation steps. This protocol has been designed for the isolation of CHO cells growing in well plates. It can easily be adapted to other samples and cell types. Contact us for recommendations.

Materials

• FluidFM micropipette (8 or 4 µm aperture, 0.3 N/m stiffness).

• PAcrAm aliquote (0.1 mg). 

• 1 ml of ultra-pure water.

• 3 ml of PBS. 

• 1 ul of 10x Trypsin solution, filtered. 

• CO2 independent growth medium without phenol red (e.g. Leibovitz's L15 medium), with 1% penicillin/streptomycin, filtered (for the already existing cell culture), serum free.

• Complete growth medium (e.g. DMEM-F12), with 10% FBS and 1% penicillin/streptomycin, filtered (for the newly isolated cells). 

• CHO cells, at 70% confluence. 

• 2x 12 well plates (ThermoFisher-Nunc). 

• Tabletop microcentrifuge and vortex mixer. 

PREPARATION 

Prepare coating solution

In order to avoid the adhesion of the cells onto the micropipette surface it needs to be coated with an antifouling material, PAcrAm. 

1. If stored at -20°C, temperate the PAcrAm aliquot at room temperature. 

2. Centrifuge the aliquot for 5 s. 

3. Dilute the PAcrAm in 1 ml of ultra-pure water. 

4. Vortex for 5 s. 

5. Dispense all the PAcrAm solution into one well e.g. A1. 

6. Dispense 1 ml of PBS in three more wells e.g. A2, A3 and A4.

Mounting and filling the probe

1. Fill the reservoir of the FluidFM micropipette with 1 ul of 10x Trypsin solution. 

2. Mount the FluidFM probe onto the system.

3. Fill the probe by applying 400 mbar until it is filled. Then switch to 1 mbar.

Coating the probe

1. Immediately after filling dip the FluidFM micropipette into the PAcrAm solution

2. Incubate the micropipette into PAcrAm solution for 30 min.

3. Wash the probe 3 times in PBS by moving into the 3 wells containing PBS (A2, A3, A4) for 5 seconds each. 

Preparation of the cells

1. Plate cells at least the day before of the experiment, so the day after will be at 70% confluency. 

2. Shortly before the experiment, remove the medium from the cells and wash them once with PBS. Add then 1 ml of CO2 independent growth medium without FBS to the cells.
   Note: FBS inhibits trypsin activity! 

3. Once the probe coating is finished, place the cells in the system. 

4. Make sure the laser signal is good and the probe is centered.

Reception wells

These plates or wells will be used to place the cells that have been picked up. 

1. Prepare each well with 1 ml of CO2 independent growth medium without FBS. 

CELL ISOLATION

Trypsinization of the target cells 

1. Approach the surface and retract 20 µm above the cells. 

2. Apply 500 mbar pressure to release 10x Trypsin solution around the targeted cells.

3. Move the probe around the desired cells.

4. When trying to detach only 1 or 2 cells, apply lower pressure (50 mbar).
   

5. Cells will slowly change their shape into a more round and bright morphology.

6. Keep applying pressure until the cells are sufficiently detached from the surface

7. Then go back to 1 mbar.
   

Cell picking

Pick up the desired cell with the following parameters:

• Approach the cell with 30 um/s and apply a pressure of -50mbar

• Setpoint 50 mV, for 5 seconds

• As soon as the probe is retracted, change the pressure to 1 mbar
  

Note:

These parameters are given as an indication, and can differ according to the experimental conditions (cell type, confluency, surface of the plate, etc).  

Cell release

Go to your target container and release the cell by using the following parameters:

• Pressure +50 mbar for 2 seconds, before touching the surface

• Back to 1 mbar

• Approach with 30 um/s to setpoint of 50 mV, stay on surface for 2 seconds

• Retract the probe by 2 um

• Move the probe laterally and fast away from cell

Confirm by eye that the cell has been released from the probe. 

Repeat

• Go back to the initially trypsinized cells. Repeat the cell picking and cell release protocol as needed. 

• Alternatively, detach cells from another area of the plate, repeating then the trypsinization step before the picking and placement.

Incubate

Once finished, incubate the isolated cells for 1 h.

After this time, exchange the medium gently, place fresh cell culture growth media with FBS to the culture and put the cells back into the incubator.