G418(Geneticin) is not killing my cells - (Mar/29/2013 )

Hello,

I've been trying to make a kill curve for my cells for more than a month now and I've been having problems killing them with G418. I'm using HPSC (Human pancreatic stellate cells ). These are fibroblast type cells found near pancreatic tumor. I used G418 from invitrogen and cells kept growing with normal speed after 2 weeks. I'm trying a new batch of g418 right now. It's been 4 days with new batch and cells keep growing with normal speed so I'm starting to freak out a little bit I'm using concentrations of 0, 100ug/ml, 300ug/ml, 500ug/ml, 750/ug/ml, 1000 ug/ml. I've read on this forum that sometimes it might take almost a month to kill cells but I was wondering what people do once they become confluent. I plate them at fairly low confluency in the begining in a standard 6 well plate, but after 4-5 days they become confluent and I start splitting them, so i was also wondering if that could effect them somehow.

I know this cells were previously selected with zyocin. is it possible that they would have resistance to g418 as well ?

Any help would be greatly appreciated!
Thanks,
Mike

Maybe you should try a higher concentration of G418. I used it on some cancer cell lines last month and killed them ..I used 0, 1mg/ml, 2mg/ml, 3mg/ml, 5mg/ml. I think 3mg/ml was effective and it is within the clinical relevant dose. Hope this helps I am only a new researcher.

I agree with Peniel. For HeLa S3 cells, I've had to use so much that the medium starts to turn yellow! Make the most concentrated solution of G418 you can directly in medium and then test a dilution series to find the concentration that kills your parental cell line and not your G418-resistant sub-cell line. It generally works best to trypsinize the cells and plate them freshly in the G418....it shouldn't take weeks as the non-resistant cells will be floating/dead the next day.