J774.2 Macrophages - Any protocols for their subculture? (Nov/22/2009 )

Hello, I am doing a science fair project that involves the culturing J774.2 macrophages for measure of cytokine release. I've been unable to find protocols for their subculture online - could anyone help me with this? I've never worked with these cells before.

I know they're a semi adherent cell line, have a doubling time of 17 hours, should be scraped when subculturing, and do best in a DMEM + L-glutamine + 10% FBS medium (I'm also using HEPES b/c I'm culturing outside of a co2 incubator).

But I'm unsure about the medium renewal, etc. How often? Should the medium just be added or do I need to replace it?
And how often should they be subcultured?

Oh, and I know this is completely separate, but I'm going to be dissolving a chemical in DMSO and then exposing it to the cells. I know cells don't respond well to DMSO in how quantities, so I'm going to be using 0.1% DMSO diluted in the culture medium. Is that a fair dilution?

Thanks - any help you could give me would be much appreciated.

Dear Prof. Moriarty,

J774 cells normally stick like superglue to any TC plastic I have ever used...if they are truly semi- adherent, there is something seriously wrong with your cells.
If you are using a closed system (Hepes) buffered, then you should top up with medium every couple of days. This allows some diffusion of oxygen into the cells. If the DMEM changes colour i.e. to an orange/yellow, then the cells need to be split/passaged/subcultured.

0.1% DMSO is the highest concentration you can use in cell experiments...above this level and cytotoxicity becomes an issue.

Hope this is useful

Kindest regards

Rhombus