General Electroporation Protocol for Mammalian Stem Cells :

Cell Density :

Cell Count: Typically, you want to start with 1–2 x 10⁶ cells per electroporation reaction.

Preparation: Cells should be in the log phase of growth. If they are too confluent, they might not electroporate well.

Plasmid DNA :

Use 1–10 µg of plasmid DNA per reaction. For high-efficiency transfection, a higher DNA concentration is typically recommended, but this can vary based on plasmid size and type.

Electroporation Buffer :

For mammalian stem cells, use an optimum electroporation buffer such as Opti-MEM, which is frequently used for stem cell electroporation to reduce toxicity and promote efficient uptake of the plasmid.

Some protocols might recommend a specific buffer that is compatible with the electroporation system you are using (e.g., Bio-Rad’s Gene Pulser or Bio-Rad's MaxCyte).

Electroporation Conditions :

Voltage : The voltage is one of the most critical parameters. For mammalian stem cells, start with a range of 250–350 V.

Capacitance : 950 µF is a common starting point.

Resistance: For stem cells, an optimal resistance is usually 200 Ω.

Post-Electroporation Recovery :

After electroporation, immediately transfer the cells to a culture medium appropriate for your stem cells (e.g., stem cell maintenance medium, such as ESC medium, iPSC medium, or MSC growth medium).

Allow cells to recover for 24–48 hours before starting selection or analysis, depending on your experimental setup (e.g., if using a selection marker, wait until after recovery to apply the selection pressure).

Time Between Pulses (if using multiple pulses) :

Some protocols with difficult-to-transfect cells recommend 2–3 pulses (10–20 ms duration each). However, keep in mind that longer pulse duration may increase cell death, so balancing cell viability and transfection efficiency is key.

Hello Binh Duong ,

It depends a little bit on your EP device. The reason why people just state they're using a "program" is that some of the popular machines will not let you set parameters, but simply choose programs.

In our case, we have a Lonza nucleofector 4D, which works really really well, that is if you have figured out the correct program for your cells. There are ways to do that.

But the programs themselves are black boxes and there's no way of telling what the machine really does. There is only the name/ code of the program. It's the same with their buffers.

However, for the Lonza devices, there is a rather large database for cell lines and programmes that work. So that's a starting point.

But then, once you've figured that out, you can achieve very high efficieny and viability.

Hope that helps.

Thomas

(no conflict of interest to declare)