Kill curve protocol specifics - (Oct/11/2010 )

Could someone suggest (link, title...) a good source providing the steps/details/protocol of a kill curve? Something I can later on cite in my thesis?

I will be using G418 on HeLa cells. G418 concentrations of 0, 300, 400, 500, 600, 700 ug/mL

Should I change the G418-laced media every day? If so, can I just make individual batches of G418 at the different concentrations and keep those for a couple weeks? Or will the G418 be deactivated if I do it in bulk like this (ex. a vial of 300 ug/mL, a vial of 400 ug/mL...)

I'm not sure what concentration of CELLS to use in the wells in which I will be subjecting them to G418

For a "kill curve," is it necessary to actually make a curve? Or just look for what concentration gives death in the 4-5 day region.

First post!
Thanks a ton

I would have a look in R.E. Freshney, Culture of Animal Cells: a laboratory manual, there is likely to be something in there.

You shouldn't need to change the medium every day, it should be changed as you would normally. You can make up individual batches, though I am not sure of the effect of heating and cooling on G418. G418 is pretty stable though, it will keep for a few months at 4 deg C.

Most antibiotics work best on cells that are less than 70% confluent, so I would start with a number that will give you about 70% confluent after several days... i.e. start with about 20-30% confluency. In 6 well plates this will be about 100,000 cells per well for most cell lines.

Many people don't physically make a curve, they just stain the plates with crystal violet or similar dye and take photos for records. To do it properly you would harvest the cells from a well each day and count to see the proportion of cells that have died compared to the controls.

Thanks for the solid answers, and I look forward to checking out that book.

Chemist on Mon Oct 11 20:19:41 2010 said: