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SCFS / Adhesion experiment (AFM Series)

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Maria Milla

FluidFM micropipettes can also be used to analyze the adhesion forces of a single cell to a specific substrate or to its neighbor cells. This protocol can be performed in all the AFM Series, and is designed for an adhesion experiment on of CHO cells growing in 12 well plates. It can easily be adapted to other samples and cell types. Contact us for recommendations. 

MATERIALS

  • FluidFM micropipette (8 µm aperture, 2 N/m stiffness).

  • 1 ul of Ultra-Pure distilled water. 

  • CO2 independent growth medium without phenol red (e.g. Leibovitz's L15 medium), with 1% penicillin/streptomycin, filtered (for the already existing cell culture) and 10% FBS, filtered. 

  • PBS. 

  • Bleach 5%

  • CHO cells, at 70% confluence. 

  • 4x WillCo wells.  


PREPARATION 

Preparation of the probe

No coating of the probe is required for this experiment. The probe must be loaded with 1 µl of Ultra-Pure distilled water. 

Preparation of the cells and cleaning plates

1. Plate cells at least the day before of the experiment in a WillCo dish, so the day after will be at 70% confluence. 

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 to the cells.

3. Prepare 3 WillCo wells for the washing sequence: 

    - Dish #1: Bleach 5%;

    - Dish #2: PBS;

    - Dish #3: CO2 independent growth medium.


ADHESION

Mounting and filling the probe

1. Fill the reservoir of the FluidFM micropipette with 1 µl of Ultra-Pure distilled water.

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.

4. Once the probe is already mounted, place the cells in the system. Make sure that the laser signal is good and the probe is centered.

5. Measure the Spring Constant: adjust the ambient temperature in each measurement.

6. Measure the sensitivity: make sure that the surface is free of cells for this measurement.


Adhesion experiment

1. Approach the surface and retract 30 µm above the cells. 

2. Move the probe on top of the desired cell. 

3. Measure the adhesion forces with the following parameters: 

    a. Setpoint: 20-30 nN;

    b. Movement speed: 1 µm/s;

    c. Pause: 5 s;

    d. Pressure: we recommend to start at lower pressures (e.g. -150 mbar) and increase the negative pressure in case it is         needed, up to -600 mbar;

    e. Retraction Distance: 30-50 µm. 

Note:

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

Cleaning of the cantilever after an adhesion event

Before measuring another cell, the cantilever must be cleaned to avoid any cellular debris to interfere in further measurements.

1. Dip the probe in the wells following the next sequence:

        1. One wash on dish #1 (bleach solution, 5%): 50-100 mbar, 30 s;

        2. Three washes on dish #2 (PBS): 0 mbar, 5 s;

        3. Three washes on dish #3 (CO2 independent growth medium): 0 mbar, 5 s.

 3. The probe is now ready for another adhesion measurement. 

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