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FluidFM micropipettes can be used to separate cells with specific properties or to avoid serial dilution procedures in isolation steps. This new protocol was released in the ARYA version 2.1.0, and is designed for the isolation of CHO cells growing in 12 well plates.
FluidFM micropipette (8 or 4 µm aperture, 0.3 N/m stiffness).
1 ul of 10x Trypsin solution, filtered.
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 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
Follow one of the coating protocols indicated in this link in order to avoid debris and cells to adhere on the surface of the probe.
The probe must be loaded with 1ul of 10x Trypsin.
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.
FBS inhibits trypsin activity!
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 plate with 1 ml of CO2 independent growth medium without FBS in each well, where the cells will be placed after the isolation.
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.
Preparation of the system
1. Select and follow the Preparation workflow in order to prepare the probe for the isolation experiment.
In case the probe has been coated inside the FluidFM system, move directly to the Maximize Signal step.
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.
Trypsinization of the target cells
1. Use the Probe Control tool to approach the surface and retract 20 µm above the cells.
2. With the Pressure Control tool, apply 500 mbar pressure to release 10x Trypsin solution around the targeted cells. Move the probe around the desired cells. When trying to detach only 1 or 2 cells, apply lower pressure (50 mbar).
3. Cells will slowly change their shape into a more round and bright morphology. Keep applying pressure until the cells are detached from the surface.
1. Select the Cell isolation workflow.
2. With the cross-hair, select the center of the cell that will be isolated.
3. Select the parameters for the isolation:
These parameters are given as an indication, and can differ according to the experimental conditions (cell type, confluency, surface of the plate, etc).
4. Click on Pick to start the procedure.
5. As soon as the probe is retracted, the pressure is automatically released to Idle (1 mbar).
6. In the next step, confirm that the cell has been picked up and is located in the cantilever aperture. If so, proceed with the next step. When the cell has not been picked up, repeat the cell picking steps.
1. Go to the new container in the right port (reception plate) by using the Navigation Tool.
2. Release the cell by using the following parameters:
3. At the end of the release process, the XY stage will perform an automatic movement of 50 um to the left, which allows the release of the cell.
4. Confirm that the cell has been released from the probe.
Use the history menu from the Navigation Tool to go back to the initially trypsinized cells. Repeat the cell picking and cell release protocol as needed.
Alternatively, use the Navigation Tool to detach cells from another area of the plate, repeating then the trypsinization step before the picking and placement.
Once finished, incubate the cells inside the system at 37°C for 1 h. After this time, exchange the medium gently, place fresh cell culture growth media with FBS to the cultures and move the cells to the incubator.
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|Asked: 4/10/19, 3:32 AM|
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|Last updated: 11/27/19, 3:07 PM|