Stable transfection selection with puromycin - (Apr/13/2006 )

Hi...

I transfected jurkat E61 cells with
1. Pbabe Puro containing ShRNA
2. Pabe puro empty

....after one day I select with puromycin (1ug/ml and 3ug/ml).. I also select E61 cells (without any construct) with puromycin as a control..

Today is the 7th days since I put puromycin but I still can see many live cells in E61 cells (without any construct) plate (control) .... Is this normal?? because I read that it ussualy takes 5 days after puromycin for the the cells to die....

I saw colonies formed in Pbabe puro with shrna plate and in pbabe puro empty plate but I can't tell if that cells contain my construct because the control plate still have many live cells....

Can I know how to differentiate live and death cells for this suspended cells (jurkat T cells )?

Thank's alott

hi
i think your puro may be out of date or not properly prepared/stored... Puro is the fastest antibiotic for selection, and 7days seems a lot. 3µg/ml seems ok. But it may be increased up to 10µg/ml (that selective con. for Hamster PC10 cells)

Puromycin selects usually within 2-3 days. So i agree with fred_33 - there might be something wrong with your puromycin stock. I'm not sure myself, but I've heard puromycin is not very stable - therefore if it was stored for long time, and not in a proper way it might have degraded and you effective concentration is lower than you think.

Live Jurkat cell - round, looks like floating ball. When the cell is dying, its not nicelly rounded, shrinks, and finally looks like it totally dried. After selection you'll have to split the cells couple of times (spinning them to remove the medium) to get rid of those completelly).