P.S. Did anyone ever try to use one of Lonza's human cell kits (e.g. CD34+ prog.) for murine cells?

I do not have direct experience with mouse progenitor cells, but from experience I have seen post-electroporation viability of human hematopoietic progenitor cell lines decrease significantly if:

1.  Cells remain in Nucleofector media ≥10 min.

2. The incorrect electroporation program is chosen 

3. Pipet transfer is too rough

(It would help if I knew the electroporation protocol you are using and any steps to optimize this)

I would recommend looking at Lonza's cell and transfection database and see if they have an optimized protocol for primary mouse cells. Otherwise you may need to look through the literature to determine what others in the field have used previously. 

Hi! How did you overcome the problem? What kind of kit and program did you use? 

Does anyone know the characteristics of the various programs, in terms of power, duration of the impulse... in order to choose the best for our cells (which are not comprised in the list of available kits)?

Thank you in advance

 I have also used Neon Nucleofactor for transfection of GFP construct into Mouse myeloid progenitor cells. Neon able to transfect the GFP construct in myeloid progenitor cells. However, as commented by Markus and Bryan, after transfection, the viability of these cells were extremely poor. Any idea to increase the viability of cells after transefction could be highly appreciated?

I used Bcl2 transgenic cells progenitors and nucleofected them in buffer V or L according to the manual with the programs X-01 or X-05. The plasmid that I am transfecting is rather large (pSpCas9(BB)-2A-GFP (PX458) from Dr. Feng Zhang's lab ~kb). In non-Bcl2 transgenic cells I have hardly any GFP+ cells. About 10 - 25 % of the Bcl2+ cells are in the life gate 24 h post nucleofection. Of those 10 - 40 % are GFP+.

I would test these conditions (Buffer V/L & programs X-01/5 individually). For me L and X-01 worked best. I also tested the human CD34+ kit with the program U-08 from Lonza, but it was not as efficient as the other buffers).

Hi! How did you overcome the problem? What kind of kit and program did you use?

I agree with Bryan J Gall about the handling of human HSCs. I spin cells slowly (100g for 10 minutes) and use transfer pipettes (wider than pipet tips = less shear stress on cells) to re-suspend cell pellets very gently, taking special care to avoid bubbles (more mechanical stress on cells). Before electroporation, I prep EVERYTHING as much as possible, to reduce time spent in Lonza's toxic electroporation solution. This includes little things like turning on the machine beforehand, labeling cuvettes and plates, warming media in the plates, making master mixes, and even pre-opening paper tabs on the little pipettes that come with the kit, so I'm not wasting time fiddling with them. Once the cuvettes are loaded, I literally run to the machine.

Regarding programs, some are definitely more rough than others, so definitely optimize by trying a panel of programs (maybe using the strips instead of cuvettes to save on cells and reagents). Their suggested optimization protocol for our instrument is here: https://bioscience.lonza.com//lonza_bs/US/en/download/content/asset/21325

We have the Lonza Amaxa 4D-Nucleofector X unit, which sounds like it might be different from your Amaxa, so look up the optimization protocol for your specific machine, or call them (I talked to "Gregory" once; he was very helpful). I have only used P3 solution for my human CD34+ cells, and looked at programs CA137, DT100, DZ100, EE100, and EO100. DZ100 was the roughest but had the highest efficiency. EO100 was a good compromise between viability and efficiency (as measured by incorporation of GFP plasmid). Like you, I am also working with a large plasmid; the Lonza control GFP plasmid that comes with their kit is only about 4kb and enters much more efficiently than the 8.5kb plasmid we are attempting to use. I will be posting my own question soon asking for tips.

Good luck!