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This protocol provides information for an efficient injection of protein into different compartments using the FluidFM technology.
FluidFM nanosyringe, 600nm 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
Make sure that all solutions (buffers and cell media) 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.
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, use a protein solution without glycerol.
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 FluidFM BIO Series system 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 1% penicillin/streptomycin.
Prepare the system
Turn on the system, the microscope and the computer.
Initialize the software.
Load the probe into the probe holder in the right port of the system.
Load the TC-treated 6-well plate containing the cells in the left port of the system.
Run the Preparation workflow.
In some cases (e.g. GFP), the filling process might take longer than expected: we suggest to follow the filling with a bright field preset. Once it is filled, move to a well which contains just medium, switch to the respective fluorescence filter and apply high pressure for few seconds to make sure the protein fills up properly all the micro channel and that there is a flow coming out from the cantilever.
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 well plate and following precisely this protocol, the parameters used should be the following:
Parameters need to be adjusted when using other cell lines or culture dishes.
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.
We have joined forces with Harvard's Wyss Institute.
Collaboration to improve CRISPR-based multiplexed gene editing.
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|Asked: 3/8/19, 10:09 AM|
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|Last updated: 6/10/20, 7:30 AM|