Nano-injection into single adherent cells
Many types of compounds can be injected into cells using the FluidFM® technology. This protocol has been optimized for the injection of Lucifer Yellow into HeLa, HEK or human iPS cells. In this case, Lucifer Yellow can be used as a potential tool to study the connection between neighboring cells in this cell lines, since it can be transferred between them through GAP junctions. A more detailed protocol is described in the following file:
FluidFM® nanosyringe, 800nm aperture, 2N/m nominal stiffness
Lucifer Yellow (2mg/ml fresh solution in HEPES buffer or TE pH7.4 buffer)
HeLa cells growing in a TC treated 6-well plate
Make sure that all solutions (including cell culture medium) have been filtered through a 0.2µm pore before starting the experiment.
The surface of the FluidFM® nanosyringe needs to be hydrophobic to avoid cell adhesion. Therefore, a thin layer of a silicone based solution (Sigmacote®) is added to the probe, following the coating protocol. The probe can be coated and stored up to one week before its use.
Prepare a 2mg/ml Lucifer Yellow solution in HEPES or TE buffer. After filtering the solution through a 0.2µm pore, dispense 1µl of this solution into the probe reservoir according to the probe preparation protocol. In order to avoid the drying of the solution into the probe, it is strongly recommended to load the probe on the BOT as fast as possible after this step.
In order to facilitate the injection procedure, it is recommended to seed the cells at least 6 hours before the experiment.
Using medium without phenol red helps to obtain a better visualization of the injection of fluorescent compounds. Therefore, follow the following steps:
Just before the injection procedure, remove the cell culture medium.
Add 2 ml of CO2 independent growth medium supplemented with 2nM of L-Glutamine, 10% FBS and penicillin/streptomycin.
Prepare the system
Turn on the BOT, the microscope and the computer.
Initialize the software.
Load the probe into the probe holder in the right port of the BOT.
Load the TC-treated 6-well plate containing the cells in the left port of the BOT.
Run the Prepare System workflow.
Once the system is ready, run the Injection workflow.
Selection of the cells
Use one of the proposed tools to target the nucleus or the cytoplasm of the desired cell. When performing cytoplasm injection, it is strongly recommended to select a point close to the nucleus. Otherwise, the probe can not enter properly into the cell and the product would be released in the cell culture medium.
Set parameters for injection
When using HeLa cells on a TC-treated multiwell plate and following precisely this protocol, the parameters used should be the following:
Parameter Value Setpoint 1µN Approach Speed 50µm/s Retraction Speed 5µm/s Pressure 30mbar Injection Time 10s Retraction Distance
Parameters need to be adjusted when using other cell lines or culture dishes.
Examples of nano-injection into adherent cells. Injection of Lucifer Yellow into 4 HeLa cells (A), 3 HEK cells (B) and 1 human iPS cell. In the case of HeLa cells, a throughput up to 200 cells injected per hour could be achieved.