antibiotic dose response for suspension cell - (Oct/21/2005 )
I am going to test the dose of the antibiotic(G418) to my suspension cell line Hmy2.C1R. This is a B-lymphoblastoid cell line. I usually culture cells between 2x105 cells/ml to 1 million cells/ml. So what cell density should i start with for testing the dose of G418. I don't know how long i need to determine G418 concentration. do i need change medium during this time? I am wondering how i can spin down cells from 6 well plate and remove dead cells due to the growth property of suspension.
thank you very much!
1. Cell seeding density: use 2-4 x 10e5 cells/ml
2. G418 incubation time: you will need to watch the cells for 1-2 weeks
3. Changing media: Easiest thing to do ... seed cells in lowest volume feasible for the well size. At day three, just add the same volume again containing double the G418 concentration (i.e. the [G418] will halve as the volume doubles). At day 6-7, add same volume again with triple the G418 concentration. Check to see cells are dying.
When you have maxed out the volume, spin cells and remove spent media and use normal volume of media-G418 to resuspend cells in well.
Use the concentration of G418 that kills cells by day 5-7 and there is no viable cells at day 14.
On most leukemic cell lines I have worked with, 500-750 µg/ml G418 works well.
When you have maxed out the volume, spin cells and remove spent media and use normal volume of media-G418 to resuspend cells in well.
Use the concentration of G418 that kills cells by day 5-7 and there is no viable cells at day 14.
thank you very much!
When you said at day 3, add the same volume media containing double G418 concentration Is that because G418 i added at day1 has already been degraded? so have to double G418 concentration when medium volume doubles and make G418 conc. in the same well always same with day 1?
Thanks!