problem with Mono Mac1 differentiation - (Aug/03/2009 )

hi,
Does somebody has experience working with Mono mac 1 cells?
I need differented Mono mac 1 cells for my experiment. In some publications, it was mentioned treatment with LPS or PMA 72h, Mono mac 1 cells should be induced to differentiation.However, I have tried to stimulate cells with PMA or LPS (different concentration from 20ng/ml to 250ng/ml) for 3 to 5 days, and cells are still floating, no adhesion anymore. I have no idea what is going on here

Thank you very much for your help!

Have you observed them from day one? Perhaps 3-5 days is to long and they start to die?

In my experiments the same cells don't really adhere when I add LPS but they cluster together. Even after 6 hours I can see the difference between 0, 0.1 and 10 ng/ml. (largest clusters in wells with 10 ng/ml)

Ytje

Ytje on Aug 5 2009, 03:14 AM said:

Thank you! Yes, I have observed them 1 day-3 day after treatment, and they do cluster together, after 1 week, almost 10% cells adhesion and the other cells are dying.
In my experiments I need monocyte-derived macrophage for HIV infection, so I decided to use differented THP1 and Mono Mac 1.For PMA-differented THP1, all cells attached and have morphological changes. In mono mac 1 case, I am not sure if cells have clustring spreading without adheasion, whether I can use them as monocyte-derived macrophage . the other thing is, is that possible to get at least 90% cells adhesion?

Thanks again for your time

I'm sorry, I don't have that much experience with these cells.
I've just done a few experiments with LPS and I really don't know enough about differentiation to help you out.

You probably read the paper from Genois et al, journal of Leucocyte Biology, 2000.
Have you tried staining some Mono macs (after 72 h LPS) for CD4, CXCR4 ect. to see if you find the same results?

Ytje