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Automated SCFS / Adhesion experiment (FluidFM OMNIUM)

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Tina Pekec
FluidFM micropipettes can be used to analyze the adhesion forces of single cells in an automated way with up to 50 cells measured in 2-3h. This protocol can be performed in the ARYA software in the FluidFM OMNIUM systems and is designed for an adhesion experiment on CHO cells growing in 12 well plates.

 

MATERIALS

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

Note: The size of the aperture depends on the size of the cell and needs optimization. 

  • CHO cells, at 70% confluence

  • Ultra-Pure distilled water (ddH2O)

  • 1x PBS

  • Bleach 14% (hydrogen peroxide)

  • 2x 12 well plates (ThermoFisher-Nunc)  

 

PREPARATION

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).


Preparation of the cells

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

2.       In this protocol, sensitivity can be tracked over time. Leave one well only with             media dedicated for sensitivity measurement if needed.

3.       Use the Exchange Plates workflow to place this plate in the left port of the             FluidFM OMNIUM system.


Preparation of the wash plate
1.    Take a 12 well plate (ThermoFisher-Nunc) and fill the wells according to the schematic:



  •  B1: 3 ml of ddH2O;

  •  A2: 2.5 ml of 14% bleach

  •  B2, C2: 3.5 ml of ddH2O;

  • A4, B4, C4: 3ml growth medium.

2.    Place the probe plate on top of the wash plate and clip securely!

3.     Use the Exchange Plates workflow to place them in the right port of the system          (keep the “probe plate” selected as default plate).

 

Preparation of the probe

1.     No coating of the probe is required for this workflow. The probe must          be loaded with 1 µl of 1xPBS.
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.

 

Note: Select the effective spring constant for your measurements. This spring constant is corrected for each type of the cantilever. You can find more details here: Why is the Effective Spring Constant important? | Cytosurge.

 

ADHESION

1.     Make sure that the probe is aligned on the magnification you plan to use. We recommend 20x.

2.     Select the Adhesion workflow.
3.     With the crosshair, select the center of the cells that will be analyzed. We recommend selecting up to 50 cells. Select cells that are far apart and are not surrounded by rounded cells or debris. The reason for this is that the cells that are close to each other will change shape if the neighboring cell is picked up. This will lower the accuracy of targeting. On the other hand, if there is something below the cantilever, there is a chance to have a false detection of the cell and the measurement will fail. The 50 cells are a limit because after a certain amount of time the cells will divide and move so the targeting accuracy decreases with the number of cells selected. 

4.     Adjust the focus on the cells.
5.     Measure the adhesion forces with the following parameters:


Note:

It is a good idea to do few adhesion measurements before starting the automated workflow. Here you can define the parameters that work best for your cell type.


6.    When measuring adhesion on multiple different cells it is important to wash the cantilever in between the adhesions to remove the cell debris. For that select the wash mode – wash after pickup:

 

a.       Make sure that the plate containing the cleaning solution is already on the             right port.

b.       Select the wash sequence:

  • B1 (ddH2O) 1000mbar 5s

  • A2 (14% bleach) 10mbar 5s

  • A2 (14% bleach) -20mbar 5s

  • A2 (14% bleach) 100mbar 5s

  • B2 (ddH2O) 500mbar 5s

  • C2 (ddH2O) 500mbar 5s

  • A4 (media) 100mbar 5s

  • B4 (media) 100mbar 5s

  • C4 (media) 1mbar 5s

c. Make sure that the “Return to current position after washing” is ticked.