Plating cells in Serum-free medium - How to plate Mcf7 cells in a serum free medium? (Jun/10/2008 )

Hi,

I am desperatly trying to plate Mcf7 in serum free medium. But whatever I do, they tend to form clusters and don't attach. I am using Krebs-Henseleit medium as a serum-free medium. And as soon as I add serum to the Krebs-Henseleit, the cells attach.
But since my experiments requires serum-free medium, I have no idea how to continue.
Does anyone has any suggestions how I could solve this problem?
Thanks a lot in advance!!!!
Bye
Franziska

If you are doing something like transfections (e.g. RNAi) try plating in normal medium first, wait till they attach and then treat. Reverse transfection (transfect and plate at same time) should in theory be more efficient, but the normal transfection works fine too.

Hi:
You have to plate the cells whith serum media, allow to attatch for 1 hour and then change to free serum media

Don`t forget to wash the serum media, at least twice whith PBS or the buffer you use

Thanks for your suggestions. The problem is that I can not change medium after plating.
I want to check the effect of some small compounds, and they have been delivered already in the plates. So I have to plate the cells in serum-free medium and then leave them in there.
I have tried different conditions and cell dissociation methods, but nothing worked so far.
Does anyone has experience with such an assay?
Franziska

there are several serum free media for tumor cells already available like quantum263 from PAA or lonza ultraculture.
your cells should attach in these media.

you can also try to adapt your cells to serumfree medium (= normal medium without FBS). start with 5% FBS and decrease every week 0.5% (there is a more detailed protocol on the lonza website).

As far as I know, cells need factors in the serum to attach. You may need to plate your cells in a fresh 96 well plate, let them attach (usually takes few hours or longer depending on cell type), and then change to serum free media as suggested. Then add your compounds to the wells - don't you want to dilute the compounds in cell media anyway? Cells won't be happy in undiluted compound solvent. Also how long will you leave your cells in serum free condition? Most cells will start to die after 24-48 h.