I am trying to transfect SH-SY5Y cells with two plasmids with a high efficient method. I have decided to use Lonza nucleofector in order to obtain better results than using FuGene or Lipofectamine. I have tested cell culture kit and PBS with 1ug or 2ug of each plasmid. In both conditions it works (also PBS!!), better with 2ug than 1ug of plasmids, but I have a high cell mortality in all conditions and the number of cells seeded was very low. Do you have any idea?
There are recommendations on the Lonza homepage for several cell lines and primary cells. I remember that they have several optimized protocols for SH-SY5Y cells and I remember that the cell viability was not that high after transfection. But I don't remember the exact numbers. Maybe you can look it up.
Additionally I can say that endotoxin is a crucial point if the transfection leads to high cell mortality. Lonza recommends to use DNA extracted with Qiagen EndoFree Maxi Kits. I don't know if there are any kits available where the purity of your DNA gets even better.
Time is a very important point. The faster you do this transfection approach and the faster your cells are back in culture medium and in the incubator the better they survive. To improve viability you can seed your cells with culture medium with only low Calcium concentration. Due to the fact that this electroporation makes holes in the membrane calcium can go inside your cell and activate different pathways. We tried this with primary neurons and seeded them with RPMI. Ca-free HBSS should work as well.
I get a high efficiency when using FuGene with my SHSY5Y cells. Maybe you can try several ratios transfection reagent:plasmid.
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