Nano-injection of proteins


This protocol provides information for an efficient injection of protein into different compartments using the FluidFM® technology

MATERIALS

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

TE buffer (10mM Tris-HCl pH7.4, 0.25mM EDTA)

HeLa cells or human iPS cells growing in a TC treated 6-well plate

UHRF1-CFP and Cas9-GFP protein solutions 

CO2 independent growth medium without phenol red (e.g. Leibovitz's L15 medium), supplemented when necessary with 2nM L-Glutamine, 10% FBS and 1% penicillin/streptomycin.

 

Warning!

Make sure that all solutions (buffers and cell media) 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.

Protein preparation

Proteins can crystallize or aggregate in the probe. This can lead to the clogging of the FluidFM® nanosyringe during the filling step. To avoid such problems, it is strongly recommended to not use protein solutions at high concentration, as well as filtering the resulting supernatant before the loading into the FluidFM® nanosyringe (using for example, centrifuge filters Ultrafree-MC-VV, Millipore). When possible, prefer a protein solution without glycerol. 

Reservoir filling

Dispense 1µl of the protein 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 1% 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 or human iPS cell nuclei, 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
Pressure75mbar
Injection Time3s
Retraction Distance

20µm

Note
Parameters need to be adjusted when using other cell lines or culture dishes. 

Perform injection.



Examples of protein injection into cells. (A) Injection of UHRF1 into the cytoplasm. The protein was fully relocated into the nucleus within 10 sec. (B) Injection of Cas9-GFP protein in Human iPS cell nucleus. 

Need more info?

This documentation page has been extracted from the Q&A section where you can discuss it and get feedback.
Related question