Dear all,
I know there are a lot of Q&A here about S2 cell culture and transfection. Believe me, I read them all. But I still have some questions.
I tried two independent transfection using Qiagen Effectene and plasmids with pAC5.1 backbone for overexpression. Same plasmid, same scale (2 ug in one T25 flask), same ratio between plasmid-enhancer-effectene. But, transfection efficiencies are significantly different since I also transfected a pAC5.1-GFP for control. The first batch of transfection, >60-70%; the second, ~10-20%. It is so weird and frustrated. The only difference between the two transfection is cell passage, first P3-4, second P8-9, I observed the cells under microscope, cell morphology does not change so much and the protocol says passage number under 50 can be acceptable...
I am new to S2 cell culture (I used to do a lot of cell culture of 293, NIH3T3 and cancer cells). Is it so sensitive to passage change on transfection rate? Because I was shocked by comparing these two transfections. Can anyone share their experience on S2 maintenance and transfection? That would be great!
Thanks,
Lu Wei
I would like to express my opinion. Even fetal hetatocyte Hc cells has already been infected at 18.6% by invaded microbes (please see file; HepG2 Fucoidan). Bacteria (Lactobacillus casei Shirota) also has been infected at 12.0% by invaded bacteriophages (my unpublished observation at National Research Institute for Child Health and Development, Setagaya-ku, Tokyo, Japan).
Although I have not determined the proteins of Insect cells yet, similar situations may be present. Interestingly, Sf9 cells have produced mainly Baculoviral proteins during biotechnology experiment (my unpublished observation at The University of Tokyo, Bunkyo-ku, Tokyo, Japan). Therefore, transfection experiment by S2 cells should be done by monitoring the invaded microbes with our PDMD (Protein-Direct-Microsequencing-Deciphering) method.
Hi!
In my experience you have always some variation in your transfection efficiancy, but between 10 and 70 % is extreme.
The best way to get variation down is standardization of every possible step and practice. The more frequently you transfect and the more standardized the protocol is, the more stable your number will become.
In general, the quality of the DNA you use for transfection is also very important. DNA quality is dependent on how you prepare it, the concentration and the storage, including freeze-thaw cycles. You should also make sure that the transfection reagents are stored according to the protocol and that they have the right temperature when you use them.
Good luck! Cheers, Tilo
You are welcome Lu Wei! Please remember to recommend answers so we get a rating, showing others that we are serious contributors ;) Thank you!
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