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HEK 293T Transfection problem


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#1 seadog

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Posted 20 August 2012 - 07:14 AM

Hi there,

I'm new to doing transfections. I have a special cells line lenti-x 293T that is an HEK 293T derivative supposedly optimized for transient transfections. I followed the protocol for GenJet online which suggests transfecting at 60-70% confluency and harvest 3 days post transfection. However, after a single day, I've noticed that there's a lot of dead cells and debris in the media, and that the cells already look overgrown and somewhat apoptotic. Should I be worried about proteases from dead cells? Is this a usual phenomenon to see dead cells?

Thanks.

#2 seadog

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Posted 20 August 2012 - 10:03 AM

Anyone have any experience with 293T cells?

#3 seadog

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Posted 20 August 2012 - 12:09 PM

Is protease ever a worry with apoptotic 293T cells?

#4 bithorax

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Posted 20 August 2012 - 02:06 PM

Change medium after transfection, different transfection agent requires different times (4h up to 16h). Those agrent are toxic to the cells (you can imagine they need to permabilize membrane in order to deliver DNA).
If you are using PEI, change medium after 6h - should help.
I also use more confluent cells: 80% - 90%.

#5 bob1

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Posted 20 August 2012 - 02:38 PM

Most transfection methods will kill a proportion of the cells, how many are killed depends on the transfection reagent to a large extent. I'm not familiar with GenJet, but there is a good chance that it is based on polyethyleneimine (PEI) with some other cationic compounds in there too, like most transfection reagents (TRs). THe presence of death does not necessarily indicate that those cells have been transfected, just that their membranes have been disrupted by the TR causing cell death.

I wouldn't be worried about the proteases in the medium, unless you are looking for a secreted protein that you hope to harvest from the medium.

293-T are extremely easy to transfect, they are one of the more commonly used cell lines for this purpose. They do have the drawback that they are only weakly attached and can, and often do, float off with knocking of the flask, pipetting directly onto the cells, and treatments with chemicals (including TRs).

Your lenti-x cells are probably best for production of lentiviral particles, rather than just transient transfections.

Many other cell lines are very good for transfection, depending on what you are doing, another cell line might be a better choice for transient expression.




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