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293T Transfection Problems/ Apoptosis measurements


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#1 bob

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Posted 08 January 2004 - 02:14 AM

Hello everybody !

Here a short description of my problem:

My 293T cells - actually a cell line which is known to be transfected very easily and efficiently (no matter by which method) - behave quite strange because control transfection with GFP e.g. look nice under the microscope + on Western Blots but my protein of interest is somehow not being expressed. Of course I checked the antibody, the plasmid DNA, the lysis of the cells, different transfection mehtods...and so on...but I can definetly rule out having any problems with that. Looking at different time points after transfection (24h, 48h) doesn´t change the problem eather.

So could please...if someone has a creative idea...write back to me.

By the way...
does anyone one know a suitable human cell line system which integrates two advantages meaning efficient transfection and being suitable for measuring expression of apoptotic markers by flow cytometry. (my passaged 293T definetly do not become apoptotic upon induction with death stimuli; they die but there´s no DNA laddering, increased caspase activity etc.)

So any way..many thanks to the people who decide to answer to me.

Bob

#2 Skeletor

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Posted 12 January 2004 - 08:55 AM

A little more information would help ;)



---Has your sample of vector DNA ever been used successfully in these (or any) cells? What type of vector is it? Is your gene under the same viral promoter as the GFP? What ratio of GFP plasmid to expression vector are you using? What % transfection are you getting?

Is a Western your only means of detecting its expression? Have you previously transfected this line with a similar vector (under similar promoter) and had success in measuring expression?


“By the way...
does anyone one know a suitable human cell line system which integrates two advantages meaning efficient transfection and being suitable for measuring expression of apoptotic markers by flow cytometry. (my passaged 293T definetly do not become apoptotic upon induction with death stimuli; they die but there´s no DNA laddering, increased caspase activity etc.)”


You did not mention which agonist(s) you have attempted.


Instead of finding the cell line that will respond to your apoptotic inducer, you may find it easier to instead find the apoptotic inducer that your cells will respond to.

There are numerous inducers of apoptosis- some are VERY cell specific while others are more general in mechanism and more probable to work in any cell. Topoisomerase inhibitors, for instance, are well established for this purpose and are popular because of (in)expense, convenience, and wide-range effectiveness. You will be able to find information on particular apoptotic agents, doses, flow protocols, etc, etc with a quick literature search for “293T” and “apoptosis.”

Here is a link to some apoptosis inducers. You likely already have some of these agents in your lab.

http://www.emdbiosci...y.asp?catid=592

Edited by Skeletor, 12 January 2004 - 08:55 AM.





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