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Nano-injection into single adherent cells

By
Maria Milla
on 3/8/19, 5:46 AM 36 views


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. 

Materials

FluidFM® nanosyringe, 800nm aperture, 2N/m nominal stiffness

Lucifer Yellow (2mg/ml fresh solution in HEPES buffer)

HEPES buffer

HeLa cells growing in a TC treated 6-well plate

CO2 independent growth medium, supplemented with 2nM L-Glutamine, 10% FBS and penicillin/streptomycin

 
Warning!

Make sure that all solutions (including cell culture medium) have been filtered through a 0.2µm pore before starting the experiment.

 

Preparation

Probe Coating

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.

Reservoir filling

Prepare a 2mg/ml Lucifer Yellow solution in HEPES 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.

Cells preparation

In order to facilitate the injection procedure, it is recommended to seed the cells at least 6 hours before the experiment.

Note

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.

 

Procedure

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.

 

Injection

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:

ParameterValue
Setpoint1µN
Approach Speed50µm/s
Retraction Speed5µm/s
Pressure30mbar
Injection Time10s
Retraction Distance

20µm

Note
Optimization of the parameters needs to be adjusted when using other cell lines or culture dishes. 

Perform injection.