Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

G418..HELP!!..I'm only a HeLa farmer!!

hTERC fibroblast G418

  • Please log in to reply
5 replies to this topic

#1 j47027

j47027

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 14 March 2013 - 11:26 AM

Hi,

I really hope somebody can help me with this.

I got fibroblast cells from another lab that are hTERC immortalised and G418 was the selective agent.
The cells were passage 15 when they arrived and I didn't add the G418 until 10 days after the I woke the cells up (I completely forgot). The cells were split 3 times and now at passage 20 they have stopped growing.

I was using DMEM with stabilised Glutamine, which the previous lab did not use.
I am also using a different LOT of FBS.

Do you think not adding the G418 after 10 days is the problem?

I really hope the passage 17, 18 & 19 frozen vials can be saved because this is the third batch of cells I got off these guys that I have killed otherwise!!!

Thanks

An embarrassed under-experienced "scientist"

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,516 posts
371
Excellent

Posted 14 March 2013 - 11:37 AM

The DMEM/glutamine should be fine, though if the other lab was using another medium (i.e. not DMEM) this may be a problem.

I presume that if the cells were made into a stable cell line, that they are not actually passage 15 (this is likely to be the passage since they were thawed or perhaps since the cell line was established) and aren't primary cells, but if they were primary cell lines then you wouldn't expect to get more than about 20-25 passages out of them (Hayflick limit).

The G418 maintains the plasmid in the cells, not adding may or may not be a problem depending on how stable the transfection was, however, as your cells seem to have stopped growing I suspect that they have lost the plasmid - and once they have reached the Hayflick limit, there isn't anything much you can do to restore growth.

#3 j47027

j47027

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 14 March 2013 - 02:19 PM

The DMEM/glutamine should be fine, though if the other lab was using another medium (i.e. not DMEM) this may be a problem.

I presume that if the cells were made into a stable cell line, that they are not actually passage 15 (this is likely to be the passage since they were thawed or perhaps since the cell line was established) and aren't primary cells, but if they were primary cell lines then you wouldn't expect to get more than about 20-25 passages out of them (Hayflick limit).

The G418 maintains the plasmid in the cells, not adding may or may not be a problem depending on how stable the transfection was, however, as your cells seem to have stopped growing I suspect that they have lost the plasmid - and once they have reached the Hayflick limit, there isn't anything much you can do to restore growth.


Thank you for your reply..

Ok so the other lab was using DMEM also...

I am a little confused by a part of your answer.."that they are not actually passage 15 (this is likely to be the passage since they were thawed or perhaps since the cell line was established) and aren't primary cells"

I would have assumed that these were primary cells that have been transfected with "immortalising plasmid" so therefore meaning that these are now immortalised...yes?

And I would also assume that these cells were considered immortalised at say passage 12-14..then these cells were split once or twice more and sent over to me. (they were sent live and not frozen)..I thought that if these cells are immortalised then they would stay immortalised as long as there was G418 in them (Which I haven't done)..

AAhhhhh..I apologise..I didnt read your answer right...

Its pretty safe to assume that these cells are not immortalised anymore then?

Edited by j47027, 14 March 2013 - 02:24 PM.


#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,516 posts
371
Excellent

Posted 14 March 2013 - 03:47 PM

To be a new cell line, the inserted plasmid should be stable - in your case, it sounds like all you had were transfected cells. The normal way to make a cell line involves transfection, selection, plating out for single colonies, screening for insert, growing the colonies up to a point where they can be frozen down. This results in a cell line where the plasmid is inserted into the genome. This usually takes quite some time and many passages, hence my doubts that the cells were actually p15.

Yes, I am guessing that your cells have lost the plasmid, adn I have doubts that it was actually stably inserted too...

#5 j47027

j47027

    member

  • Active Members
  • Pip
  • 11 posts
0
Neutral

Posted 14 March 2013 - 04:03 PM

To be a new cell line, the inserted plasmid should be stable - in your case, it sounds like all you had were transfected cells. The normal way to make a cell line involves transfection, selection, plating out for single colonies, screening for insert, growing the colonies up to a point where they can be frozen down. This results in a cell line where the plasmid is inserted into the genome. This usually takes quite some time and many passages, hence my doubts that the cells were actually p15.

Yes, I am guessing that your cells have lost the plasmid, adn I have doubts that it was actually stably inserted too...


Ok thank you..I am going to have to get the specific details from the other lab on how they generated this cell line.

But one more thing..

Do you think its possible for a cell line to adapt in such a way that it will only grow in one specific LOT of FBS?..that was the only other thing i could think of that may have been the problem. They have grown and generated all their cell lines in the same batch of FBS over the course of the last few years.

Edited by j47027, 14 March 2013 - 04:05 PM.


#6 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,516 posts
371
Excellent

Posted 14 March 2013 - 08:03 PM

Batches of FBS can vary a lot, so yes, it is possible for the cells to grow slower or look a bit different in different batches of FBS. I don't think that ordinary MEF style fibroblasts would be so finicky that they would only grow in one specific batch of FBS. It may pay to find out where they get their FBS from. For example if they use New Zealand FBS and you are using some from Europe, you might well see some quite big differences.





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.