4D Nucleofector for Transfecting Human Fibroblasts

January 20, 2016

BlueskyReddit
Harvard Medical School
Neurobiology
Postdoctoral Fellow

Overall

Performance

Ease-of-Optimization

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Company:

Lonza

Product Name:

4D Nucleofector X

Catalog Number:

AAF-1002B/AAF-1002X

We are interested in knocking out mutant genes using CRISPR/Cas9 technology. Patient-derived cells provide a relevant model to assess CRISPR action on disease-causing genes. When screening for selective guide RNAs, we transfect the CRISPR plasmids into fibroblasts using the 4D Nucleofector device. Fibroblast transfection is not easy, but high performance of the nucleofector system allows excellent transfection rate - yet the optimization of transfection remains a challenge.

Experimental Design and Results Summary

Tips

There are some tips that will definitely help to improve survival. (1) Use endo-free maxi-prepped plasmid DNA, (2) Pipette cells gently, particularly after nucleofection to prevent cell death, (3) Stick to low speed (90g) centrifugation, (4) make sure your cells are not infected with Mycoplasma

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Summary

The Good

It works, fibroblasts are hard to transfect with other method. No other method was superior to Nucleofection in our hands.

The Bad

Quite expensive, not easy to optimize. The company protocols are not very helpful in optimization. They don't tell you the likely best protocols.

The Bottom Line

It's a great tool to perform transfection, but the user needs some experience and optimization; it is time consuming.

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