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C2C12 differentiation and transfection


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5 replies to this topic

#1 laurequillo

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Posted 04 March 2010 - 11:40 AM

Hi there!

I am working with C2C12 cell line, using medium with 2% horse serum to induce differentiation. The problem is that this cells grow like crazy, so if I want to transfect them and then induce the differentiation for some days the "control" is already too grown! And when these cells overgrown they differentiate as well. So the point is, how you do it? You seed different amounts of cells and use the low density cells as a control and the other to do the transfection and differentiate?

Any suggestion is welcome!
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#2 giulin-giulÓ

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Posted 27 July 2010 - 07:30 AM

Hi!

I am working with C2C12 too....and I am really getting crazy!
I am sorry that I didn't reply before, but I just signed in.

I spent the last few months in transfect them without any results...did you manage to do it?
Did you use Lipofectamine 2000 to transfect them? When I used it I had the same problem because I had many dead cells in may samples, but not in my control (without siRNA).
I don't think that plate a different amount of cells is a good idea...I guess that you wanna see how the transfection influence the differentiation process and if you plate different amount of cells how can you compare with the control?

Unfortunately, mine are just suggestions....I don't have any good results so far :(
Shall we share some info about these bloody cells????


Thanks

#3 laurequillo

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Posted 27 July 2010 - 07:40 AM

Hi!

I am working with C2C12 too....and I am really getting crazy!
I am sorry that I didn't reply before, but I just signed in.

I spent the last few months in transfect them without any results...did you manage to do it?
Did you use Lipofectamine 2000 to transfect them? When I used it I had the same problem because I had many dead cells in may samples, but not in my control (without siRNA).
I don't think that plate a different amount of cells is a good idea...I guess that you wanna see how the transfection influence the differentiation process and if you plate different amount of cells how can you compare with the control?

Unfortunately, mine are just suggestions....I don't have any good results so far :(
Shall we share some info about these bloody cells????


Thanks


well, it is good to see that I am not the only one... Now I have more info and some results;

I transfect the cells with rotifect, and the efficiency is quite good. They dont die with the transfection (I saw that they start to die after maybe 5 days differentiating)
For silencing I use lentivirus, with good results as well.

The point is to use enough cells to get the transfection, but not too much to avoid the differentiation, and once you start the differentiation process it is ok. Once I start the differentiation I freeze the sample without DM, so they wont grow anymore, and later you can check the amount of cells, but normally is not so different (maybe from day 3 on you will have more, but you can adjust the levels)

You should use a control like a scramble plasmid, or an empty vector. Another thing is to use plasmids that give you a marker to select the cells (i.e. puro), then you will get better results.
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#4 giulin-giulÓ

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Posted 27 July 2010 - 08:02 AM

Great!!! I am happy that you did it!

I use siRNA to knock down my protein, not lentivirus...but how many cells do you use for the transfection? I heard that it's easy to transfect them when they are not so many. And...how many days after the transfection do you put the DM?

#5 laurequillo

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Posted 27 July 2010 - 08:10 AM

Great!!! I am happy that you did it!

I use siRNA to knock down my protein, not lentivirus...but how many cells do you use for the transfection? I heard that it's easy to transfect them when they are not so many. And...how many days after the transfection do you put the DM?


I dont count the cells, but I seed from one big flask with 20ml, and I use 1ml per plate.
I transfect them, and I wait 1 or 2 days and I add the DM. (At that poin I collect the Day 0, without DM)
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#6 laurequillo

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Posted 29 July 2010 - 10:19 PM

I guess that you wanna see how the transfection influence the differentiation process and if you plate different amount of cells how can you compare with the control?


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I seed the same amount, and actually once they start differentiate they dont grow so much (they stop proliferating)...if you run a gel, in the tubulin you can see that day0, day 1, day 2 have the same amount. At day 3 you have to maybe use more volume to do the lysis, but not big deal.
"He must be very ignorant for he answers every question he is asked" Voltaire

"This is SPARTA!"

"I┤m the goddamn batman"




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