Bone marrow transplantation - (May/14/2005 )

Dear all,
I'm working on hematopoietic stem cells (HSCs) and I'm trying desperately to perform retrovirally mediated mouse bone marrow chimeras. The technique has always been working in our lab but recently problems started accumulating. I isolate HSCs from donor CD45.2 mice, infect them with retro or lentiviruses and then inject them in CD45.1 lethally irradiated hosts. Has anyone got some tricks to help me, a reliable protocol or some troubleshooting hints?

what are your problems with the bone marrow transplantation ?
are your cells not growing?
the only problem we have had with our bone marrow dendritic cells
were that they were growing horrible on our corning dishes and experienced significant decrease in cell populations from day 0 to harvesting day.
we swtiched to 6 well plates and i get 86 percent recovery on harvesting day.

I'm actually not trying to isolate bone marrow dendritic cells, but I'm doing long-term repopulation assays of stem cells... I do not culture my cells, I isolate them from a mouse, manipulate them (viral infection) and then inject into a lethally irradiated mouse, and then check for engraftment a few weeks later...

Thanks anyway...