I am working with adaptively transferring leukocytes in tumor-bearing mice. So I am taking the cells from the donor animals, label them with fluorescent dyes and inject them back to the recipients. However, because of all the unnecessary damages that labelling of the cells in vitro causes, I am looking for a way to label the neutrophils in vivo.
Currently I am planing to label the monocytes in vivo so I am also thinking to label the neutrophils as well in different settings.
Moreover, I wondering when I label the circulating blood monocytes in vivo how many monocytes I can label and how many of them actually migrate to the tumor xenograft!
The question you raise is challenging. First how can you get a large number of neutrophils for labeling. We found that we needed at least 10e7 to track trafficking. Getting cells from a tumor bearing animal is likely to result sin MDSCs rather than PMNs. Indeed, even if you inject G-CSF many of what you might call neutrophils could be progenitor cells. Genetically labeled cells are good although we have had success with CSFC labeled cells as well. Similar problems would occur with monocytes. Easier to do with BM differentiated macrophages.
Please see Int Immunopharmacol. 2012 Jul;13(3):245-56. Tumor regulation of myeloid-derived suppressor cell proliferation and trafficking. Younos IH1, Dafferner AJ, Gulen D, Britton HC, Talmadge JE. where we did this using CFSE labeled cells.
James @ Thanks a lot for the tips. I have read your nice paper and I got some ideas for my work!
I was thinking to use the same method fir isolating Gr+ cells. However, I am wondering of using BrdU, shall I also inject to the donors as well? So when I am collecting them from the spleen of TB donor mice, they are positive.
And then should I inject BrdU to the recipients?
You have used CSFE in your nice work, however, I need a second colour as for labelling due to my work, in which I am comparing the trafficking behaviour of Gr+/Gr- in two different recipients.
I have been using CMTPX, which is red, but I have found this colour is as stable as CSFE. So I am going to use CMTMR, which I think is orange. Do you think this colour is as stable as CSFE?
I used tumor primed BM to examine/compare their homing to the tumor, but unfortunately I don't get that much cells out of BM so I was thinking to use the same population from spleen as I think I would get much more cells out than BM, what would you think?
You can use BM cells that you expand and differentiate into macrophages. Its a standard technique. This will provide large numbers of cells for study.
Regarding I was thinking to use the same method fir isolating Gr+ cells. However, I am wondering of using BrdU, shall I also inject to the donors as well? So when I am collecting them from the spleen of TB donor mice, they are positive. I am not sure how normal BrdU labeled cells would be. I assume BrdU labeling would induce mutations. BrdU labeling provides a way to assess cellular proliferation and does not address bio-distribution.
And then should I inject BrdU to the recipients?
You have used CSFE in your nice work, however, I need a second colour as for labelling due to my work, in which I am comparing the trafficking behaviour of Gr+/Gr- in two different recipients. As you point out below lots of ways to label cells.
I have been using CMTPX, which is red, but I have found this colour is as stable as CSFE. So I am going to use CMTMR, which I think is orange. Do you think this colour is as stable as CSFE? I have not used CMTMR. You will have to read the literature and qualify the approach.
As I am studying MDSCs and their trafficking profile in my WT vs. KO mice, I am going to enrich MO-MDSCs and PMN-MDSCs from spleen of TB mice and inject to recipients. Would you think that it would be better to enrich them from BM or spleen is also ok? (as the characteristics of splenic MDSCs and BM-MDSCs are different).
Also I am thinking to study T-Cell trafficking cells enriched from spleen to the tumor in my WT vs. KO mice.
BM will give you macrophages. M-MDSCs are found in low numbers in the spleen and other organs. We were never able to get enough to study bio-distribution. To get around this problem we labeled total spleen cells and used flow to identify who went where. However, still not enough M-MDSCs to track. We found you needed about 10e7 labeled cells to track over time. The use of CSFE lets you follow cell proliferation as well.
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