Macrophage culture problems! please help! - macrophages (Apr/21/2005 )

I am currently trying to culture macrophages isolated from salmon head kidney on a percoll gradient. I am seeding them out in 24 well plates letting the cell suspension adhere at 15 degrees for 3 hours and then washing with 0.1% FCS L-15 media. the aim of the experiment is to infect the cells with virus and then look at the antiviral response over 9 days. However i am yet to infect with virus as i am getting a crash in numbers after about 3 days. I am replacing media every two days (maintance media L-15 supplemented with 5% FCS). After 3 days it just looks like there is nothing in the wells and it isnt even worth doing counts on the haemocytometer as basically there is nothing there. Iam not wanting to use antibiotics as they will interfere with the pathogen and possibly also intere with the immune response of the cell which is what i am looking at. Can anyone help or provide some helpful tips for keeping macrophages alive for atleast 9 days. Time is running out for my PhD so i am desperate!

Thanks,

Gavin

Hi Gavin:

First, have you checked the medium's pH?
Second, why do you incubate these cells @ 15 degrees, why not 37?
Third, have you considered to let cells attach during 6-8 hours?

Good luck!