How to do transfection on 293 cell - (Aug/09/2005 )
Hello, everyone:
I am doing transfection on 293cells. You know this cell line is easilly detached from the plate. However I have to change to SF media when do transfection as the only transfection reagent availabe now is lipofectamine. So far I have tried several times and everytime I change media very gently in order not to distrurb the cells. But still a lot of cells come off from the plate. Obviously, this cause the transfection efficiency is different from sample to sample.
In addition, I use about 40% confluence to do transfection by following a paper. I know this confluence is lower than normal transfection and there is a lot of cells dettached. But the results(I am doing luciferase assay) looks ok, though not perfect. I also use 80% confluence by following some other papers and there is not too much cells decttached. But the results is bad.
Now I really do not know which one I should follow.So is there anyone have any experience on tranfecting 293???
To help adhere the cells I coated my slides in poly-L-lysine. It worked great for keeping the cells from coming off the slides. Here's the protocol I used:
Prepare poly-L-Lysine (PLL) solution and coat a 4-well chamber slide under sterile conditions in a laminar flow hood.
1. Dilute PLL 1:10 with PBS
2. Add 400μl 1:10 PLL solution to each well.
3. Allow to coat for 60 minutes.
4. Wash once with 750μl PBS.
5. Allow to dry for approximately 3 hours. The slides can be stored indefinitely at room temperature under conditions to maintain sterility.
As mentioned I used 4-well chamber slides so I didn't need much volume at all for coating the slides but you can scale it up to whatever you need.
When I transfect 239-T cells, I go like this: on monday I plate about 700.000 cells in a 60 mm dish.
The next day, in late afternoon, I use CaPO4-method with about 12 µg of DNA to transfect the cells, and on wednesday morning I gently remove the medium and gently add new medium, and almost no cells detach. The way to do is, is to take the plate and hold it at a small angle (not horizontal) and just take of the medium and add the fresh medium slowly (while the plate is still not horizontal). Then put it back to a horizontal position and back in your incubator.
As I do transfections to produce virus, I harvest on friday, and get great yields.
The next day, in late afternoon, I use CaPO4-method with about 12 µg of DNA to transfect the cells, and on wednesday morning I gently remove the medium and gently add new medium, and almost no cells detach. The way to do is, is to take the plate and hold it at a small angle (not horizontal) and just take of the medium and add the fresh medium slowly (while the plate is still not horizontal). Then put it back to a horizontal position and back in your incubator.
As I do transfections to produce virus, I harvest on friday, and get great yields.
Thank you for your guys reply!
So Vairus metioned that 700,000 cells is plated on the 60mm dish. So the confluence when doing transfection is about 60% to 90% right? So it seems that high confluce give good transfection??
In addittion, poly-L-lysine will affect the transfection ??
Hello,
As for 293cells transfection, i also coat my glass coverslips with poly lysine.
Yet, i use another transfection reagent, which is very convenient compared to lipofectamine.
Its name is Fugene6 (Roche Labs ; http://www.roche-applied-science.com/fst/p..._5_3_18_1_4.htm ) : no need to use serum free medium, no cytotoxicity so you can leave the reagent in the medium until you analyze your cells.
I use 12mm diameter glass coverslips in 24 well plates (nunc), coated with poly l lysine.
24h before transfection, i plate about 50-60000 cells in each well in DMEM supplemented with 10% fetal calf serum. In these conditions, the confluence % is usually 50-80%.
Then, for the transfection : 2,5 - 3 µl fugene6 for each DNA microgramm.
hope this will help