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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 ARYA software in all the BIO Series, and is designed for an adhesion experiment on of CHO cells growing in 12 well plates.
FluidFM micropipette (8 µm aperture, 2 N/m stiffness).
1 µl of Ultra-Pure distilled water.
CHO cells, at 70% confluence.
2x 12 well plates (ThermoFisher-Nunc).
Initialization of the software
1. Turn on the microscope and the fluorescence lamp. Turn on the system control unit and the pressure controller. In the computer, open ARYA.
2. Create a new project and 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
No coating of the probe is required for this workflow. The probe must be loaded with 1 µl of Ultra-Pure distilled water.
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. 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. Use the Exchange Plates workflow to place this plate in the left port of the FluidFM BIO Series system.
4. Prepare a 12 well washing plate with the following the next distribution:
- B1: 2.5 ml Bleach 5%;
- A2, B2, C2: 2.5 ml PBS;
- A4, B4, C4: 2.5 ml CO2 independent growth medium.
5. Once the probe has been loaded into the system, use the Exchange Plates workflow to place this plate in the right port of the system (keep the “probe plate” selected as default plate).
Preparation of the system
1. Select and follow the Preparation Advanced workflow in order to prepare the probe for the isolation experiment.
- Measure the Spring Constant: adjust the ambient temperature in each measurement. Click on Measure, and once the plot and the values appear, apply the results and save the data for further analysis.
- Measure the sensitivity: make sure that the surface is free of cells for this measurement and that the approach speed is set up at 10 µm/s (Settings > BOT). Click on Measure and, once the value has been showed, on Apply. In case the value can’t be measure, use 1.5x10-6 as a default value (by clicking on “empty”).
2. In order to align the probe, approach it to the surface of the well with the Probe Control tool and drag the crosshair to the center of the aperture.
1. Select the Adhesion workflow.
2. With the cross-hair, select the center of the cell that will be analyzed.
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.
4. Once the measurement is done, check the results on the Results View (folder icon, on the bottom left corner of ARYA).
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. Make sure that the plate containing the cleaning solution is already on the right port.
2. Select the washing tool (laundry icon, on the left side of ARYA) and follow the next sequence:
1. B1 (2.5 ml of bleach solution, 5%): 50-100 mbar, 30 s;
2. A2, B2, C2 (2.5 ml/well of PBS): 0 mbar, 5 s;
3. A4, B4, C4 (2.5 ml/well of medium): 0 mbar, 5 s.
3. Once the sequence is ready, click on play and the system will start the cleaning.
4. The probe is now ready for another adhesion measurement.
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|Asked: 8/6/20, 4:19 AM|
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|Last updated: 8/6/20, 9:48 AM|