Recently I have some studies on Myeloid derived suppressor cells (MDSC). In my opinion, they mostly look like the granulocytes and monocytes. Firstly, how can I distinguish MDSC from common granulocytes and monocytes?
More importantly, we are trying to knockdown some of the genes in MDSC. The MDSCs are harvested by flow cytometry sorting from tumor bearing mouse. But how can I culture them? The life of granulocytes should be just around one days, but it may take at least two days for the transfection and gene knockdown. Is there anyway to culture them in vitro for 2 or 3 days? From some paper, I try to add some GM-CSF, IL4 and IL6 into the medium. The cells look survived, but I just don't know if they are still MDSCs.
I'm just a newcomer on this field. Still taking effort on the literature. Thank you everyone for any suggestion.
GM-CSF or IL4 will keep these cells viable. but they will also regulate the suppression potential of MDSCs.
Hi Junhua!
MDSCs is a new field of research.
I have only worked with MDSCs from peripheral blood of human patients with breast cancer. By flow cytometry you can distinguish granulocytes and monocytes cell populations with surface markers, either in human and mice.
I suggest you a perspective from a Nature Review Cancer, about this topic (MDSCs) that show a historical progression of MDSCs in the literature. http://www.nature.com/nrc/journal/v13/n10/full/nrc3581.html
If you need it, I have the full text.
I hope my comment helps to clear up some of your questions about MDSCs.
Best regards
Another paper that has free full text from the same author: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587148/
There is a table entitled: Phenotypic definition useful for separation of different myeloid populations, and one of the paragraph is about the typical phenotype of mouse MDSCs.
May I ask from which tissue you isolated these MDSCs?
If they were sorted from tumor tissues or spleen directly, my suggestion is to add some conditioned medium of the tumor cell or tumor tissue, to your MDSC culture. The volume of such supernatant should not exceed 1/3 of total culture media to minimize cytotoxic effects.
Or if you sort the MDSCs from bone marrow, maybe it is better to induce bone-marrow cells to MDSCs using tumor cell-conditioned medium plus GM-CSF and IL-4 after the gene is knocked-down.
To my experience, addition of tumor-conditioned medium will prolong the survival of MDSCs without changing their morphology significantly. However, MDSCs are heterogeneous and should be further identified by their T cell-suppressive function. If you are not sure whether the survived cells are still MDSCs, why don't you try to co-culture them with activated T cells, and determine the T cell proliferation (e.g by CFSE labeling) or IFNg production?
If you need further information about the experiment procedure, welcome to contact me and a previous publication of mine may also help. (http://www.ncbi.nlm.nih.gov/pubmed/23064360)
Good luck!
Thank you so much to you all, Asif Amin Dar, Ithaisa and Lijie!!!
Really appreciate for your great suggestions!!!
I'll read the papers in detailed.
- Badges
- Science method