Sincerely...

I would skip Nucleofector...

Is there any specific reason I may have missed why you want to use Nucleofector?

Now there are plenty of transfection reagents that can deliver highly efficiently your plasmids to the cytosol. Either based on liposomes or in nanoparticles...

From Lipofectamine to Jet Prime, etc.

Have you already tried them? They may be a bit toxic for some cell types, but it's worth trying.

There are some other low-toxicity reagents such as HappyFect, Safectine, etc...

Much luck,

Loren

hI Marcela,

before going to electroporation and have to try conditions almost from scratch by varying volts, time etc, or changing transfection method totally from electrical to chemical, which are suggested before, I would suggest you the following, on the method you are trying..

-even if the program you use is the one for the fibroblasts, I would ask them for other programs to try, and perform a short experiment trying the combination of nucleofector buffer and program... I don't know which is the nucleofector 2b, but for the nucleofector II you have two buffers to choose and several programs for optimization per cell type...

-the trick with nucleofection is to not have cells waiting on the nucleofection mixture of buffer and dna for more than two minutes BEFORE nucleofection... they wait better AFTER nucleofection... so if you are preparing several cuvettes at a time, i would prepare cell pellets separated, add the mixture and transfer to the cuvette and immediately to the nucleofector and pulse.... then you can let the cuvettes after the pulse wait for a bit, before you harvest them to pool them in same well... as

-i assume you need many millions of nucleofected cells per condition and per experiment, and crowding the cuvette is not givin you better results... so if recommended cells are half a million or a million per cuvette and nucleofector reaction, that is the reaction you need, but you need many to set up an efficient plate for reprogramming...

I expect these tricks to work for you , as they worked for me with even more delicate cells than fibroblasts...

then to seed them onto gelatin instead of nude plastic, could help them thrive...

I tried lipofectamine 3000 to generate iPSC from fibroblast cells, also very familiar with Neon transfection system. Based on my experience, if use electroporation method to delivery vectors, cell density is very important for the viability. Poor cell density should be avoided. Other than cell density, ROCK inhibitor need to be added to help cell survival.  Lipofectamine is also a good choice, I got good results from some lines.

Thanks for the feedback, all. I was able to successfully establish 12 cell lines using your suggested techniques to improve viability, including seeding them at a higher density. Now, I am transfecting the hiPSCs I created with CRISPRs using Stem Cell Nucleofector Solution 2 (Lonza), and facing similar viability issues. I suppose it is just a caveat to electroporation in general, regardless of cell line, there will be high cell death.