3 replies to this topic

[rrn]

I have generated stables with puromycin res construct which contains a gene coding for a chemokine of my interest. I need to use the spent media to culture other cells in it ! I guess the puromycin contained in the spent media is not allowing the new cells to grow. Is there any way to remove puromycin ? Is it ok if I do not add puromycin for few days and use that spent media ? Will the stable expression get lost ?

Thanks

[bob1]

Typically stable cell lines only require a maintanence level of antibiotic to keep the stable expression, so you should be using about 1/10th of the original dose.  I don't know of any way to get rid of the puromycin that won't also damage the medium components and probably your chemokines too.

It should be OK to culture for a day or two without the puro, but you would have to assay this for yourself.

[drwho]

what I have done in the past is 24/48 hours prior to you needing your media containing your chemokine remove your antibiotic containing media and replace with your normal media (with no antibiotic).  Collect that media (24/48 hourse later) and use as required, then replace with your selection media.  If the cells are stable a few days out of section should not affect them too much

Edited by drwho, 28 September 2012 - 07:31 AM.

[rrn]

yea thanks ! I removed puro pressure for 2 days and checked for expression of the gene and protein.. its there.. So i guess 24-48 hours without pressure wont affect ..

Thanks again !