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Injection of Lucifer Yellow in 50 HeLa cells and volume measurement
-FluidFM® nanosyringe, 800nm aperture, 2N/m nominal stiffness, coated with Sigmacote in situ.
-Lucifer Yellow solution (2mg/ml in HEPES or PBS or TE pH7.4 buffer).
-HeLa cells, plated in 12-wells plate (Thermo Fischer, Nunc).
-CO2 independent growth medium, supplemented with 2nM L-glutamine, 10% FBS and penicillin/streptomycin.
II. Initialisation of the software
-Switch the microscope and the fluorescence lamp on.
-Switch the BOT and the pressure controller on.
-Open the ARYA software.
-Create a new experiment.
-Align the axis if necessary.
-Select 12 well plate on the left port (set content height at 1 mm if you are not using the recommended plate).
III. Preparation of the plates:
Prepare 12-wells plate with cells
Seed the HeLa cells at least the day before.
Just before the experiment, change the medium with 1ml of filtrated CO2 independent growth medium.
Note: Adding to much medium might increase the background in epifluorescence mode.
Also prepare 1 well that does not contain any cells with 1ml of the same medium as seen in the picture.
Place the plate on the left side port. (use e.g. the exchange plate workflow to go to the left side port)
When using the exchange plate workflow, set content height at 1 mm if you are not using the recommended plate.
Prepare the loading plate:
Prepare 1 well with 3ml ethanol and 6 well with 3ml PBS as described below. This will be use in step IV.7 of this protocol. DO NOT USE POSITION A1, A3, C1 nor C3 as they are used for loading the FluidFM probes.
Place the holding plate on top of this well plate and place everything in the right port of the BOT (use the Exchange plates workflow to move to the right port).Page Break
III. Preparation of the Nanosyringe
- Coat the nanosyringes as explained in the silicone coating SOP on the online documentation.
Note: To avoid condensation during this process, use desiccant in the oven and in the desiccator.
Prepare a 2mg/ml Lucifer yellow solution in HEPES buffer.
Filter the solution through a 0.2µm pore.
Dispense 1µl of this solution into the probe reservoir according to Probe preparation instructions.
Load the probe in a free loading port of the loading plate previously prepared and positioned in the right port.
IV. Preparation of the system
-Select the Prepare system workflow.
1. Choose Probe
- switch to brightfield (choose from preset)
- choose the 10x objective
- If a probe is mounted on the measurement head, drop on an empty port
Make sure that the slot you are about to drop to is empty.
- Pick the probe you have previously prepared and positioned in the loading plate.
- Do not select the auto alignment option
- Click ok to start the probe dropping and picking procedure
- The system stops right before the gripping operation.
- Focus on the cantilever
- Put the probe to be gripped in the center of the blue rectangle (either by dragging the stage or configure and applying discrete steps)
[Text Wrapping Break]- Press OK to start the probe exchange process.
2. Check tightness of the pneumatic circuit
- Launch the flowsensor’s software “Sensirion USB RS485 Sensor Viewer”
In the field Sensor, select SDP 6xx Series and press OK and Run
- Apply 20mbar pulse using the pressure tool and check the flow in the Sensirion application
ATTENTION: Remind to remove the pressure or the probe may fill without you noticing
- If the reported flow shows more than +/- 0.02 Pa while the oscilloscope shows 20mbar, it means that the probe is not properly connected.
- If the tightness test fails, repeat the probe- picking operation. If a tight seal cannot be achieved, discard the FluifFM probe and contact support.
3. Fill Probe
- Select Fill probe from the prepare system workflow
- Select the preset probe filling.
- Focus on the probe
- Press the set new reference button
- Set the pressure to 300mbar
- Apply filling pressure (10s to 1min)
- Wait for the contrast of the microchannel to change and click release pressure.
- Set the desired pressure by clicking on the value and changing it. 300mbar is usually enough and liquid in the cantilever can be seen within 10s after applying the pressure.
- In case any air bubble appears inside the nanosyringe, make sure to apply pressure long enough to make it disappear. Increase the pressure if necessary.
Note: the filling procedure can also be supervised in fluorescence mode.
4. Immerse the FluidFM probe
Wash the probe to avoid formation of air bubbles:
- Use the Wash tool
- Create a washing sequence using the well prefilled with PBS and Ethanol (Step II. Of this protocol):
To add a new wash step to the sequence: click on a well in the plate view and drag it to the sequence.
To change the pressure or duration of a wash step: first select the step you want to modify, then click on the property you want to edit.
- Wash first 2s in PBS: A2.
- 2s in Ethanol: B1.
- 5 times 2s in PBS (B2, C2, A4, B4, C4).
- Start the wash sequence by clicking the play icon.
5. Align Laser
- Move to your desired sample or well with the help of the Navigation tool. Here move to the well containing medium but no cells (A3).
- Focus on the probe.
- Remove the Infrared filter slider below the microscope’s objectives.
- Select the align laser preset.
- Align the laser using the align laser section in the prepare system workflow.
The laser must be aligned with the tip of the probe for the force feedback to work.
Click on “Align laser” and wait that the laser reach its reference position (picture Before alignment)
Use the arrow navigation control in the dialog to adjust the laser beam such that it is deflected by the probe’s end as shown in the right picture (After alignment)