I have been transfecting a dendritic cell line for about a year now with about a 15-30% transfection efficiency that I would like to increase to 40-50%. I have used Neon Transfection (electroporation) and chemical transfection (JetOptimus) and changed multiple parameters for the electroporation and the chemical transfections with no success. My largest plasmid is ~6.8 kb and is being co-transfected with GFP at ~4.5 kb.
I noticed you said dendritic cell line, there is a general trend that the more differentiated a cell is (i.e. neuron, cardiomyocyte etc), the harder it is to transfect. I worked with 3T3 fibroblats, HeLa cells and HL-1 atrial cardimyocytes and I can tell you that HeLa had the highest transection efficiency, 3T3 had modest efficiency with HL-1s requiring extensive transfection optimization. There are several parameters you can optimize which will enhance efficiency, I will point you to my PhD thesis online pg. 135 onwards (https://research.bond.edu.au/files/36135827/Can_Kiessling_Thesis.pdf). We ended up peaking at around 50% GFP positive cells and that wasn't really sufficient for our purposes. Whereas HeLa cells had 70% efficiency without any optimization. I would also urge you to check for mycoplasma contamination, people are generally unaware that mycoplasma contamination is very prevalent and a subtle type of contamination that is not like your typical bacterial/fungal type of contamination where you will see signs of infection i.e. pH changes in the media, growth in the media etc. Mycoplasma is a known inhibitor of transfection.
You can try to use a lentivirus to increase your transfection efficiency. You should determine the optimal Multiplicity of Infection (MOI) for your cell line and then transduce your cells.
Hi Lauren Bracken
I see your plasmid size is high and that's why instead inserting GFP inside your plasmid (Not to exceed the sized more that can be linked with multiple issue even cell death) you are co-transfecting the cells with GFP plasmid.
Now, there is something missing in your question, how much of your plasmid you use along with how much of GFP concentration, here for sure you will use higher concentration of your blind plasmid and a little of GFP just to get a little signal of transfection. If this is the case, then 15 to 30% transfection is quite fine because your blind plasmid would be more in number as compare to the lower amount/concentration of GFP that is co-transfected with your plasmid, so ideally more of your plasmid would have been transfected as compare to lower amount of GFP plasmid.
If this is the case and let say, you use 1 ug of total both plasmid (100 ng GFP+900 ng Your plasmid) or (300 ng GFP + 700 ng Your Plasmid) in 24 well plate, just have a try to use the total amount of GFP plasmid alone to mimic your original condition, you will get good transfection because there will be only GFP (in total) that is responsible for the signal, and hence you can have a comparison justification for actual transfection (Your plasmid+GFP).
If you can let us know the amount of plasmid that you are using (Both your plasmid+GFP plasmid).
kind regards
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