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
- - - - -

Cell culture medium instead of Nucleofector solution??


  • Please log in to reply
3 replies to this topic

#1 autophagator

autophagator

    member

  • Active Members
  • Pip
  • 27 posts
0
Neutral

Posted 15 June 2010 - 05:57 AM

Hi.
In our lab we have Human Cancer Stem Cells derived of pacients and I need to introduce a forgein DNA into them, but all methods fail to tranfect primary cells. So, I will use the Nucleofector Technology, but I have not the expensive nucleofector solution...
I would like to know how I can replace the expensive Nuclefector solution to electroporate my ES cells. Can I use cell culture medium of Cancer Stem Cells?


thanks in advance.

#2 krisztina

krisztina

    member

  • Active Members
  • Pip
  • 15 posts
1
Neutral

Posted 08 July 2010 - 02:15 PM

Hi

I use amaxa nucleofection a lot and my experience was that some cells don't care much about which solution I use, whereas there is a dramatic effect with others. Therefore I recommend you to stick to the solution that works best even if it is the expensive one from the supplier. Try it, though with other reagents, it might work. My primary cells seem to be less fuzzy than the cell line I use, so you never know. And my cell line did not like the recommeded buffer, I had to test various options.
Just a few of my experience with nucleoporation:
My cells are extremely sensitive to antibiotics after nucleofection, try and avoid it.
I nucleofect up to 4 million cells in one reaction that makes it cheaper (be careful not to exceed 10% volume with the DNA, though).
After nucleofection I dilute my cells in media with 20% FCS and 3 hrs later I add some more media or change media.

Hope it helps. Good luck

#3 revex

revex

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 22 March 2011 - 01:38 PM

Hi

I use amaxa nucleofection a lot and my experience was that some cells don't care much about which solution I use, whereas there is a dramatic effect with others. Therefore I recommend you to stick to the solution that works best even if it is the expensive one from the supplier. Try it, though with other reagents, it might work. My primary cells seem to be less fuzzy than the cell line I use, so you never know. And my cell line did not like the recommeded buffer, I had to test various options.
Just a few of my experience with nucleoporation:
My cells are extremely sensitive to antibiotics after nucleofection, try and avoid it.
I nucleofect up to 4 million cells in one reaction that makes it cheaper (be careful not to exceed 10% volume with the DNA, though).
After nucleofection I dilute my cells in media with 20% FCS and 3 hrs later I add some more media or change media.

Hope it helps. Good luck


Krisztina,
Have you noticed a reduction in transfection rates with increased cell count using Amaxa nucleofector at 4 mil cells?
Do you transfect Neuroblastoma cell lines? I have been using 2 mil but the price on Amaxa has gone up significantly.
Thank you for your feedback.
Revex

#4 tcheh

tcheh

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 30 March 2011 - 03:00 AM

I tested several conditions for Amaxa nucleofection for HUVECs, which are quite sensitive cells, and not many other transfection methods work well for plasmid transfections. Using medium or PBS instead of amaxa solution did not work, most cells died after the transfection.

But the following worked: Diluting amaxa solution 50/50 with PBS or medium (or probably anything else). Cells were fine and transfection rates were apparently the same for all tested plasmids. So far no problems with that approach, and it definitely saves us money.




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

©1999-2013 Protocol Online, All rights reserved.