I though it would be interesting to see what people use to transfect their common cell lines, (e.g. CHO, HEK, COS-7) and then have a discussions on pros and cos of various techniques.
I have found the liposomal delivery systems such as Lipofectammine 2000 and the newer generation reagents from Dharmacon and BioRad to be excellent for transient transfections of a variety of constructs - from large plasmids to RNAi. For stables - atleast for small constructs- the age old Ca-precip methods have worked best for me, though it seems to be no good for large constructs. Next on line is nucleoporation: lets see how it works. Will update in a few weeks!
Lipo2000 works great on neurons
Tried it on superior cervical ganglia and had nice results
I've been very pleased with Effectene for transfecting cell lines such as SKN (neuroblastoma), CHO, MCF-7 and CV-1. You use a smaller amount of DNA as compared to some other reagents. However, I have found that I need to optimize for each cell line (usually I use less effectene than Qiagen recommends, which ends up saving me $).
I thought this board deserve to know the following info (copied from another forum):
"Just want to share with this board that our lab has done a poll on current transfection methodology in a variety of cultures in the course to find a quick, reliable and cheaper mean when dealing with a particular cell line or related cell types. This info was derived from 17 phD-level cell biologists in our lab, each one has at least 3 years of cell culture experience and at least used 3 different transfection methods. The purpose is that you don't need to waste a lot of time and money to try every method and to have a more standardize procedure...
What we found is that:
for the most research purposes, the liposome method being the most popular. The first line of choices are Lipofectamine2000, GenePORTER-2, Fugene-6 or GenCarrier-1/2 and with GenCarrier the most cost-effective.
The second line of choice (e.g. if not successful with those above reagents, the next method you may consider) are adenoviral, letiviral or baculoviral systems and with adenoviral being the most cost-effective.
The last choice will be electroporation, nucleofector or immunoporation (the most recnt method on market) which requires bigger investment on instrument, optmization and training.
The above results are just our poll from our lab, not representing any formal or official results..... and just for your reference."
I agree totally with the previous post.
For cell transfection, our experience is that the new comer, Gencarrier-1, probably is the most effective lipo-reagent for common cell lines as well as hard-to-transfect lines. It outperforms lipo2000, fugene, geneporter, genejuice, jetPET, superfect, effectene in our lab (CSHL, we have different people use different reagents) on transfection efficiency, extremely low cytotoxicity and lowest price. Gencarrier now is our standard lipo-reagent for cell transfection, if it can not do the job we will switch to viral infection or other mechanic means - electroporation, nucleofection etc.....Don't waste your time and money on the other lipo reagents.
Recently, I heard many good comments about Gencarrier cell transfection reagent. I have some doubt so decide to test it myself and surprised to find that this reagent outperforms lipofectamine2000 and fugene-6 on my C2C12 myoblasts. I am really impressed with its performance especially its lower price ($180/mL) and wondering how much money Invitrogen ($260/mL) and Roche ($350/mL) make out of a small vial???
I can not help to think probably brand name is made primarily from OUR money not quality. Something to think about......someday you'll be a Lab head with your own budget.
We do a lot of large scale transfections using HEK 293 cells to produce AAV vectors and have found that calcium phosphate is still the best and since we make the solutions ourselves - it's the cheapest. The most critical factor is the type od 293 cell being used. There seems to be an inverse correlation between the level of adherence and maximal transfection efficiencies. Also the cells must NEVER be allowed to become confluent during passaging. Transfection efficiencies of over 90% are possible if you are careful, but consistently high levels are difficult to achieve.
PEI is both inexpensive and very efficient
It will however be necessary to experiment with different ratios and concentrations of DNA to PEI to optimize for each particular cell type.
I use PolyFect/SuperFect... best for most cell lines like HEK293. Cos7, U2OS, SAOS2, WI38.
Fugene6 is ridiculously expensive.
I always used electroporation for CHO and it worked wonderfully for me. I am surprised to see only 15 % votes for it.
We use lipofectamine 2000 from invitrogen to transfect lung epithelial cells and it is very efficienthttp://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect&productID=11668019&messageType=catProductDetail
Hi. I would be really interested in trying Lipofectamine 2000 onto Superior Cervical Ganglia cells. I read you tried it already. May you kindly send to me the protocol you used? I know sympathetic neurons undergo physiological modifications upon different culture conditions. Could you describe me exactly the SCG culture conditions you used and where they come from (either rat or mouse)?
I USE LIPOFECTAMINE!
THERE IS CERTAINITY IN THAT YOU WILL GET SUCCCESFULLTRANSFECTION
Even though high efficiency is obtained, what is the reason for poor consistency with HEK293 cells? Does it ahs to do with AAV you were working or with the method?
I am debating whether to use CaPo4 or Lipofectamine or the Gene carrier for transfecting 293T cell.
It is quite easy to get a 60% efficiency when transfecting 293 cells. Some would even say that you can get this by placing the DNA tube close to the petri dish! But seriously, getting efficiencies in the 80-90% is important when using the cells to make rAAVs because you are co-transfecting (usually) two types of plasmid, a helper and a vector plasmid and you really want all of the cells to get both plasmids and ideally in equal molar ratios.
Cells that have been allowed to become over confluent do not yield good transfection efficiencies, even many passages after they had become stressed. I think this is the real reason why people prefer to use low passage cells. Split them 1:10 every 3 days and you will keep them "happy".
The reason why I used CaPo4 not Lipofectamine 2000 is because when making very large AAV preps, you need grams of plasmids and a lot of transfection reagent. Since I didn't own a significant share of Life Technologies (Invitrogen at the time), I could not afford to use Lipofectamine.
Hope that helps
I am looking for a reagent that will give me very high transfection efficiency in HeLa cells. I have tried Effectene, Fugene and Genejuice but they never give me anything beyond 40%. Have you used GenCarrier-1 for HeLa cells? Do you get about 70-80% efficiency? Suggestions are welcome.