Was there something you absolutely needed to change in the general protocol to succeed? I would really appreciate some insight from people with first hand experience.
I did a test today with fibroblastic primary cells, that are difficult to transfect and we upgrade the level by doing Lipofectamine LTX 4H after platting the cells with 15µl of lipofectamine in a 12 well plate seeding at 150000 c/w with 2.5µg YFP plasmid around 30% of the cells are transfected the other way is to transfect directly at the seeding moment. not already try
Try using no antibiotics / antimycotics in your media, we have found this to be very toxic to the cells during transfection. Additionally, reverse transfecting (forming the lipo:DNA complexes in the wells, then adding your cells of interest still in suspension) always seemed to give better transfection efficiencies. Best of luck!
I have some experience with difficult-to-transfect type of cells. You may need to perform "optimization". See pg. 160 or 5.3.3 Transfection optimisation:
http://epublications.bond.edu.au/cgi/viewcontent.cgi?article=1195&context=theses
Thanks for the tips, here is what we have learnt in the past few weeks:
-3ul Lipofectamine 3000 for each 12 well is sufficient for primary hepatocytes that are freshly plated.
-It is critical to only let the DNA- P3000-Optimem mixture incubate for max 5 minutes before adding the Lipofectamine-Optimem mixture.
Hope it helps any future readers.
Hi,
Let's see:
1. 3*10^5 cells / 12 well
2. We transfect right after plating, then again in 2 days
3. No coating, but cell culture plates have special treatment by the manufacturer.
4. We prepare the transfecition mix in OptiMem, then add in to wells that already contain normal hepatocyte medium.
5. You should be able to detect GFP 48 hours after transfection. For other GOIs, I suspect it is gene-dependent.
6. Efficiency was greatly influenced by the plasmid used (the smaller the better), and was remarkably reduced, compared to control transfections in hepatocarcinoma cell lines or HEK293Ts: Less than 10% in primary hepatocytes vs 65-70% in cell lines. This efficiency is a lot less than stated by ThermoFisher, and probably has something to do with the fact that viability is greatly reduced in the transfected wells. For the record, endotoxin-free plasmid preps seemed to have no advantage, but we still used those.
Hope this all helps, but if you are looking for higher transfection rates then you will probably need to find another way. Let me know which direction you decide to go. :)
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