hygromycin selection problem - (Apr/09/2008 )

I titrated mouse hybridoma cells in 0 to 1000ug/ml hygromycin to find out optimal concentration for drug selection. After one week, about 20% of the cells were still alive even at the highest drug concentration (actually, there were about the same number of live cells in 100 to 1000ug/ml hygromycin). It looks like hygromycin does not kill the live cells, it just prevents cells from multiplying.

My question is: Is this normal? Do I have to try higher concentration or wait longer?

I haven't used mouse hybridomas but yes, hygromycin does not always kill all the cells. Try a longer incubation time and ensure you are replacing the spent media on a regular basis (say every 3rd day) to keep the antiobiotic "fresh".

You could try a higher concentration but then this may cause interesting mutations to arise in your cells or you may just clone out a fast growing, resistant subset of cells.

Hope this helps,

AussieUSA.