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Transfection contamination?


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12 replies to this topic

#1 proteinrunner

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Posted 23 March 2009 - 12:39 AM

Hi,

I was doing 3 days cell transfection in serum-free medium. I did that alright for previous times. But recently the cells always died exactly just after 2 days. It's really annoying because I couldn't see any trace of it on the 2nd day, but the 3rd day cells were all rounded up and dying. Medium was not clear but maybe it's because of floating cells. The strange thing is not all transfection dishes were bad, but, like 5 out of 8. I wonder if it is contaminated or the cells just died for some other reason? And when can that be?

I really hope you can share your experience or give any advice. Thanks!!!

#2 cotchy

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Posted 23 March 2009 - 04:16 AM

Hi,

I was doing 3 days cell transfection in serum-free medium. I did that alright for previous times. But recently the cells always died exactly just after 2 days. It's really annoying because I couldn't see any trace of it on the 2nd day, but the 3rd day cells were all rounded up and dying. Medium was not clear but maybe it's because of floating cells. The strange thing is not all transfection dishes were bad, but, like 5 out of 8. I wonder if it is contaminated or the cells just died for some other reason? And when can that be?

I really hope you can share your experience or give any advice. Thanks!!!



What method are you using for the transfection?

Lipofectamine, electraporation?

Many of these methods are lethal to cells (at certain concentrations or voltages) as the way they function is to open cell membranes (to allow in DNA) so if the membrane cannot close after it has been opened the cell will die.

Is the concentration or timelengths of your method different than the other times when it worked?

If so this could be the problem.

Cotchy.

#3 proteinrunner

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Posted 23 March 2009 - 04:29 AM

What method are you using for the transfection?

Lipofectamine, electraporation?

Many of these methods are lethal to cells (at certain concentrations or voltages) as the way they function is to open cell membranes (to allow in DNA) so if the membrane cannot close after it has been opened the cell will die.

Is the concentration or timelengths of your method different than the other times when it worked?

If so this could be the problem.

Cotchy.


Thank you Cotchy. I was using Lipofectamine. The method was exactly as before, so I don't think this can be the reason. There might be other things that did the dirty job...

#4 cotchy

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Posted 23 March 2009 - 04:42 AM

In my experience of using lipofectamine, the cells can be lethally injured by lipofectamine but this may not appear to have killed them for a couple of days at which point they 'ball up' and float.

If the medium is not cloudy you could almost certainly rule out bacterial contamination and possibly fungal as I imagine you would see this through the microscope

I have (luckily enough) not experienced mycoplasma contamination, which you cannot see visually through the microscope or media cloudiness but this does effect the physiology of the cells

However im not sure if it kills them so you could maybe even rule this out (although some others might have suggestions on this)

If we rule out contaimination it comes back to the methodology or the technique.

Was it the same cells you used each time when the transfection worked and didnt work as each cell type has different efficiencies, if they were the same cell type was the passage number significantly different as i know passage number is very imporatant in cytotox tests and so i pressume it would also be important when treateing the cells with lipofectamine.

What procedure did you use, if you have the patience to type it out i can have a look over it.

Another pair of eyes might spot something you cant.

Cotchy.

Edited by cotchy, 23 March 2009 - 04:43 AM.


#5 proteinrunner

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Posted 23 March 2009 - 05:40 AM

In my experience of using lipofectamine, the cells can be lethally injured by lipofectamine but this may not appear to have killed them for a couple of days at which point they 'ball up' and float.

If the medium is not cloudy you could almost certainly rule out bacterial contamination and possibly fungal as I imagine you would see this through the microscope

I have (luckily enough) not experienced mycoplasma contamination, which you cannot see visually through the microscope or media cloudiness but this does effect the physiology of the cells

However im not sure if it kills them so you could maybe even rule this out (although some others might have suggestions on this)

If we rule out contaimination it comes back to the methodology or the technique.

Was it the same cells you used each time when the transfection worked and didnt work as each cell type has different efficiencies, if they were the same cell type was the passage number significantly different as i know passage number is very imporatant in cytotox tests and so i pressume it would also be important when treateing the cells with lipofectamine.

What procedure did you use, if you have the patience to type it out i can have a look over it.

Another pair of eyes might spot something you cant.

Cotchy.



Ok, here it is:

Cell type was the same as when last transfection worked, and it's Px+24 and last time it was like Px+21.

And transfection procedure was as described for the Lipofectamine 2000. I used one tube for optimem and lipofectamine for 5min RT incubation, another tube for optimem and oligo. Then I combined these 2 tubes after this 5min incubation, let it stand there for around 30 min. In the end I add the mixture to antibio-free medium with suspension cells. After 4h, I change the medium to serum-free medium and wait for 3 days.

Can you see anything wrong there?

I made a new transfecion again last Saturday, so today is the 2nd day and the cells are just fine. I am really afraid to see them die tomorrow and I had a stupid nightmare yesterday that they died again.

#6 stardust

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Posted 23 March 2009 - 08:25 AM

hi,

do you always grow this cell type in serum free medium? Which cells are you using? Which serum free medium? How did you prepare ypur DNA? Could it contain endotoxins? When it worked did you use the same construct? Maybe what you are expressing is lethal?

Stardust

#7 cotchy

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Posted 23 March 2009 - 08:43 AM

hi,

do you always grow this cell type in serum free medium? Which cells are you using? Which serum free medium? How did you prepare ypur DNA? Could it contain endotoxins? When it worked did you use the same construct? Maybe what you are expressing is lethal?

Stardust



I cant see anything off hand wrong with the protocol however i recently came across a post here on the forum about using serum when using lipofectamine 2000,

A transfection worked for me in the presence of serum with lipo 2000 and also for a good few other people on the post so you could try it with serum present

Stardust makes a good point about the expression of lethal genes or endotoxins.

I pressume all other points stardust makes are the same as before when it worked?

So fingers crossed for tommorow, if it doesnt work perhaps try with serum present

Cotchy.

#8 cotchy

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Posted 23 March 2009 - 08:45 AM

Sorry i just read your post again.

Why do you remove the serum for 3 days?

Normally the transfection procedure occurs in serum free medium, for the 6 hours or so the lipofectamine is with the cells, then complete media is added to the cells for 3 days to allow the cells recover

The absence of serum (and the growth factors it contains) from your media for 3 days could be lethal to your cells

Cotchy

Edited by cotchy, 23 March 2009 - 08:53 AM.


#9 proteinrunner

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Posted 23 March 2009 - 12:12 PM

Thank you stardust and Cotchy. I'm using a cancer cell line and RPMI 1640 serum-free medium is for experiment need - it's not a problem for the cells to grow. And oligos or plasmids are not lethal for sure as previous transfections were no problem.

Now the problem is, I just checked the 2 days transfected cells under 40x objective and found there might be yeast contamination in my control oligo transfected cells. Yes it's creepy. But a lab mate who have used the same oligo now seems have not any contamination problem, but he uses DMEM. I wonder if the medium matters?

#10 WOW

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Posted 24 March 2009 - 05:33 PM

Thank you stardust and Cotchy. I'm using a cancer cell line and RPMI 1640 serum-free medium is for experiment need - it's not a problem for the cells to grow. And oligos or plasmids are not lethal for sure as previous transfections were no problem.

Now the problem is, I just checked the 2 days transfected cells under 40x objective and found there might be yeast contamination in my control oligo transfected cells. Yes it's creepy. But a lab mate who have used the same oligo now seems have not any contamination problem, but he uses DMEM. I wonder if the medium matters?


I am sorry for your situation. I don't mean to offend you but are you sure you cells are maintained in a good status. When I began to do transfection (lipo or electro), my advisor always reminded me keeping my cells always in the best status, otherwise everything would be wrong. Since every procedures seemed OK, if your plasmid are prepared by commerical kit (most of them could discard endotoxin), and your gene is not lethal, you'd better think about your cell status. You could use cells cultured by others for a trial.

May it help you and good luck.
Je pense, donc je suis

#11 memo

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Posted 25 March 2009 - 08:01 AM

Hi, I know that culture medium could be of influence to show a contamination! You probably use antibiotics or fungizone (amphotericin :) in one and maybe not in the other?
Also yeast prefers a certain media probably as some of the cancer cells I transfect die in DMEM and grow rapidly in RPMI!

Good luck! Hope your cells have survived in your last experiment!

#12 proteinrunner

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Posted 26 March 2009 - 05:22 AM

Thank you guys! I've found out the problem is the siRNA oligo. It have been contaminated. So I just discard it and buy a new one. Hopw things will be well soon.

#13 cotchy

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Posted 27 March 2009 - 01:22 AM

Sorry to hear that but at least its sorted now.

Good luck in the future

Cotchy.




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