clonogenic assay of primary cells - problem with obtaining colonies (Aug/28/2009 )

hi everyone,

i have been doing clonogenic assay of BPAEC (primary cells) after irradiation but i always get very low plating efficiency, only 5% (which is not considered to be reliable). does anyone happen to know how to improve this? i did another try yesterday, this time adding conditioned medium but since this whole thing takes 2 weeks, i'd like to try other options soon (in case adding conditioned medium fails).

thanks-
radioactive

The radiation activates p53 dependent apoptosis of the cells. You could try reducing the amount of radiation the cells are receiving.

radioactive on Aug 28 2009, 09:29 PM said:

What is the plating efficiency of your untreated (not irradiated) cells? Based on that, you can decide what needs to be improved in your assay. Maybe your cells don't do well at low density, then you need to seed more. Is attaching to the plate an issue after irradiation? Try increasing FBS % or coat your plate with poly lysine?
What is the threshold considered reliable for plating efficiency?

Well, hope that help! Let us know what worked for you!