optimizing selection of transfected cells - (Aug/28/2006 )

Dear friends,

In most of the protocols, it is suggested that after adding the media plus antibiotic to the cells for selcting the transfected cells, we should change the media every 2-3 days. but my culture media turn yellow very fast in less than 24 hours and get overpopulation. what should i do. should i still change the media every 2-3 days? (i think i have tried a reasonable range of hygromycin which is 50-250 microgram per ml).

thanks,
Maryam

---

Subculture your cell line in lower density, ie less cells per ml.

---

Dear Minnie Mouse my seeding density is very low (X200 dilution of the original flask) However the cells are very fast growing and a selection time of 2-3 weeks is too much for them. Also i think the main problems is that the dead cells can not detach from the plates very easily and i get a population of dead cells attached to the plate which makes the condition bad for the surviving colonies and ocupying the surface. Do you think trypsinizing when changing the media could be a good idea?

---