I am trying to establish intra-hepatic bone marrow transplant protocol. I have to use intra-hepatic injection in new born mice pups. in order to just have hand on the injection, the new born pups were irradiated with sub-lethal dose (which is already standardized) following which, pups (which are CD45.2) are injected with total bone marrow cells from CD45.1 mice (approx. 100000 cells). after 6 weeks of engraftment, when I sacrifice the mouse to look for percentage engraftment in mice, what I found is that there is really high engraftment in spleen (around 70%) but really low in bone marrow (less than 10-15%). Please find the attached FACS file. What can be the reason for this ?
you could try multicolout flowcytometry to look at different lineage markers (CD3 or CD4/8, B220/CD19, MAC1,...) and whether you also find a difference in chimerism then.
Maybe the overall difference is caused by different representation of various cell lineages in SPL/BM and the chimerism within a lineage is conserved.
Maybe the intrahepatic way of transplant is related somehow with splenic engraftment rather than BM engraftment. The flow charts look clear. We performed several experiments with splenectomized mice to test CXCR4 and VLA4 mobilization. The procedure was very easy and well tolerated to the mice. Maybe if several of your experiments show the same data, you could try to splenectomize the mice before the intra hepatic BM transplant. If you need I can find the splenectomy protocol
Dear Ragnav Oberoi, Nina Pilat is right, to look and analyze the total chimerism is not enough. You should check the chimerism for every cell (sub)population (granulocytes, monocytes, lymphocytes, etc). In the "normal" BM you will find more granulocytes and less lymphocytes. In the spleen will be the other way around (a lot of lymphocytes and erythrocytes and almost no granulocytes).
It is very well can be that there is no problem with the HSC (BM) homing and/or engraftment, but there is a problem with HSC renewal or HSC mobilization (due to the irradiation effect in the "young" BM niche).It is also possible that the "first environment" (liver) has an effect on the HSC differentiation (may be more towards lymphoid lineage)
Another suggestion, choose more time-points. Six weeks is too short to say something about the donor HSC (BM) activity. You should wait longer (at least 12 weeks). First weeks after BM transplantation you will mostly see the progenitor (MPP, HPC) cell activity. We (usually) take a little bit of peripheral blood from mice every 3 weeks after transplantation (3, 6, 9, 12, 15, 18) and do FACS analysis of chimerism not only for total leukocytes, bur also for the major cell subpopulations (granulocytes, monocytes, B cells, CD3+CD4+ and CD3+CD8+ lymphocytes).
I hope this will help. Success!
Thanks everyone for your suggestion. I will try to look for further population subset as well, and also give more time for engraftment. How blood do I need to have sufficient cell number for FACS analysis ? For me, FACS analysis from blood does not always work the best. Main reason is incomplete lysis (I use ACK cell lysis solution, warm, 10 min at room temp.). the blood should be first stained with antibodies and then lysis ? or lysis first followed by antibody staining.
Thanks for your suggestion once again.
Dear Raghav Oberoi,
We first lyse RBC (lysis buffer = 8.4 g/L NH4Cl and 1.0 g/L KHCO3, pH 7.2-7.4). Per sample we use circa 4 ml of lysis buffer (4C). We lyse the cells 10 min (at 4C). After these we wash the rest of lysis buffer and cell debris away and stain the cells.
First time (3 weeks after BM transplantation) we take really a little bit of peripheral blood (PB) - 20-30 ul. At this time point your mice are weak and it could "kill" the mice, if you will take bigger volumes (we use not more than 50 ul). At this time point we stain only for leukocytes (because it will be not enougth of blood to stain for all cell populations).
Later (6, 9, 12, 15, 18 weeks after BM transplantation) we take more PB (50-100 ul) and after RBC lysis stain for all (leukocytes, granulocytes, monocytes, B and T lymphocytes) cell (sub) populations.
P.S. you can also stain your cells first and then lyse RBC, but in this situation you will need to use more Ab.
Success!
Also, if you have the ability, consider being more selective with your bone marrow. Evaluating engrafement using Lin-cKIT+Sca+CD150+ might be more useful in that these are the least differentiated cells. Like Marina said, whole bone marrow has a number of short term and multi-potent progenitors that will become myeloid lineage (which might explain your spleen results).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line