In Vivo cell proliferation - (Apr/14/2005 )
I was hoping someone could provide advice on in vivo cell proliferation. In my protocol, I obtain spleen cells from a donor mouse, label them with CFSE and then transfer them to a reciepient mouse via intravenous injection of the tail vein. After 2/3 days I obtain spleen cells from this mouse and detect on the flow cytometer. As of yet, I have not detected any intensity in these cells. I have tried increasing the label, increased the concentration of cells transferred and I have harvested and checked lymph nodes and thymus. What am I doing wrong?
I am not familiar with this experiment but do you think 2/3 days is enough for engraftment?
We have been doing similar experiments, and I can share some of what I've figured out. First of all, if the recipient mouse is not irradiated or otherwise immunodeficient, the CFSE labeled cells will be like a needle in a haystack. Try an immunocompromised mouse and see what happens. Also, collect lots of events during flow (like a million or more). CFSE labeled cells can be less that 0.05% of total cells. What concentration of CFSE do you use? Can't really over-do it, as the cells will release any excess and there is a natural drop of the first day or so after labeling. Good luck
I think that 2/3 days are too much. I looked after 1 day, and detected 0.5% CFSE+ cells. I injected 5.000.000 labeled cells.