5 replies to this topic

[cellgene]

Everybody tells me something different. My ex-supervisor even was not counting and just doing it  

How many 293T or Hela cells should I seed for transfection in 10cm dishes (I want to harvest after 48h)?

And something else just came to my mind:
I know that if you have a selective medium with antibiotics (puromycin, g418 etc.), you shouldn't use the antibiotics in the medium when you are seeding cells for transfection. Is it the same for penicilin/str.?


thanks

[killmusic1979]

A good guide for the number of cells to plate can be found in the Fugene 6 protocol at

http://www.roche-app...rt/1815091a.pdf

This recommends between 1 x 10^5 to 3 x 10^5 cells for one well of a 6-well dish to transfect the following day -  so scaled up you'd be aiming for a ballpark figure of 1 x 10^6 cells for a 10cm plate. This works fine for most cell types (including HeLas), however 293Ts are very small so if you want them ready to transfect the following day I would aim to double or even triple this i.e. 2-3 x 10^6 cells for a 10cm plate. Depending on how long you transfect for they may end up very confluent by the time you do the experiment, but transfecting at lower density doesn't work as well. In my experience the confluency won't affect most experiments too much anyway.

Hopefully this helps!

cellgene, on Apr 8 2010, 09:48 AM, said:

Everybody tells me something different. My ex-supervisor even was not counting and just doing it  

How many 293T or Hela cells should I seed for transfection in 10cm dishes (I want to harvest after 48h)?

And something else just came to my mind:
I know that if you have a selective medium with antibiotics (puromycin, g418 etc.), you shouldn't use the antibiotics in the medium when you are seeding cells for transfection. Is it the same for penicilin/str.?


thanks

[killmusic1979]

Oh, also yes it's the same for Pen/Strep. The transfection reagents allow the antibiotics into the cell meaning that you'll get toxicity. However, this is dependent on the transfection reagent, I have done it with Fugene and not had a problem, but it is a problem with some others such as Lipofectamine 2000.

cellgene, on Apr 8 2010, 09:48 AM, said:

Everybody tells me something different. My ex-supervisor even was not counting and just doing it  

How many 293T or Hela cells should I seed for transfection in 10cm dishes (I want to harvest after 48h)?

And something else just came to my mind:
I know that if you have a selective medium with antibiotics (puromycin, g418 etc.), you shouldn't use the antibiotics in the medium when you are seeding cells for transfection. Is it the same for penicilin/str.?


thanks

[labrat612]

One quick point to mention, when seeding cells, it would depend on the transfection agent you are using. Some prefer a higher confluency, while others are slightly different.
Check the manufacturer's recommendation for hints on it.

[cellgene]

Thank you both.

We are doing transfections mostly with polyethylenimine and I think there is no user's manual for it

[labrat612]

cellgene, on Apr 8 2010, 12:07 PM, said:

Thank you both.

We are doing transfections mostly with polyethylenimine and I think there is no user's manual for it

I've used PEI for a few of my transient transfections as well. To start my optimization experiments, I based it off the manual for Fugene HD. The cell densities that they recommend for their product did work out well for my cell line. You could try it for yours as well.


Hope that helps!