At the workshop a lot of people mentioned using PEI as the transfection reagent for mammalian cells as well as insect cells. What supplier do people use for buying PEI?
Also, I wanted to try out some of the Thomson flasks that Sabine mentioned for insect cell growth, but forgot to write down the name of the company supplying them in Europe. . .
Thanks in advance!
For the PEI, I am using this one: http://www.polysciences.com/Catalog/Department/Product/98/categoryId__283/productId__1577 and it is probably this one others are using as well, as it is promoted by original seminal publications by both Durocher and Wurm groups (the gurus of 293:). Two years ago, the price for 2 g was around 150,- EUR in my country.
I am not aware that somebody is using PEI for insect cells but it could possibly be used...?
We just bought the same reagent from Polysciences. I am sure it needs some optimiation to get it to work, but could somebody give us some hints about a good protocol to use, when it comes to ratio DNA:PEI and amounts of DNA to use for the transfection?
Thanks Ondrej! I think there was someone at the workshop who mentioned PEI with insect cells, so I thought I would test it (I forget who it was!). I agree with Malin, it would be great if someone could post a protocol. I will check out the original publication as well.
I would suggest that you may start with 1 ug of DNA per 1 million of cells at a cell density 1 million per mililiter, that is 1 ug of DNA per mililiter of culture - one can find DNA concentration used at transfection expressed in both terms, as ug of DNA per number of cells or as ug of DNA per milliliter of culture of a given cell density, I am using the first expression as it is a more universal one - and within this standard conditions, you should try DNA:PEI ratios from 1:1, 1:2, etc., up to 1:8 or so - this could be easily done i.e. in 24- or 12- well plate with 1 or 2 ml culture in each well, should be done in triplicate and checked for GFP fluorescence level on one side and for recombinant expression level by some assay, i.e. for secreted alkaline phosphatase by colorimetric reaction. Once you have optimized DNA:PEI ratio, then you can try what amount of DNA is sufficient to transfect your cells completely - you will keep the DNA:PEI ratio constant at the previously found optimum but you will vary the amount of DNA added per number of cells / volume of medium used for transfection; you may find out that i.e. you do not need 1 ug per million but just half of that amount, so you could spare the plasmid, the transfection reagent and also the cells, for the more PEI you add to them, the less viable they will be after transfection... Hope these suggestions help, shall I try to write a protocol out of it? May be some other people should write their opinions here before and then we can have some conclusion...?
The direct contact to thomson is Sam Ellis Sam Ellis email@example.com
Concerning PEI protocol, we use the protocol published in the Durocher Chapter of a protein expression book. If you are interested, I have to get back the book (borrowed to a scientist) and let you know the details.
The protocol published in this book (I also have this book) is also available on-line at http://cshprotocols.cshlp.org/content/2008/3/pdb.prot4977.full along with four other related 293 protocols and some less related protocols, just see the "related protocols" section at the bottom of the page... Enjoy;-)