I would like to study the effect of a treatment on the infiltration and phenotype of T cells in the pancreas in NOD mice using FACS. I am not sure what the best method for processing the tissue is.
Should the pancreas be digested with collagenase? Are all CD45- cells depleted before FACS analysis? Is it necessary to further isolate the islets before FACS?
I am curious why you don't use immunohistochemistry. Obviously, it is easier and more economical to operate.
I will be doing this in addition to immunohisochemistry. The main reason for adding flow cytometry is to do more complex phenotypic analysis of T cells.
Depends on your assay set up. You could just grind the pancreas using two frosted glass slides. The downside is that when you stain, most of your cells would be pancreatic cells and you may have to acquire a whole bunch of cells to see the infiltrating T cells. To avoid this, do collagenase digestion and isolate islets using a microscope. You could try depleting CD45- cells prior to FACS but the risk is that you may end up with a very low yield unless you have severe insulitis. I guess at the end of the day, you may have to try the different approaches and find the one that works for you. I have linked a couple of papers that use one or the other.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052054#s4
https://www.cell.com/cell-metabolism/pdf/S1550-4131(17)30040-2.pdf
Julie Joseph Thank you for the response. I was able to digest the pancreas with collagenase P and detect CD4 and CD8 T cells within the pancreas. However I am now wondering if a perfusion to removed the blood before hand is critical. If this is not done will most of the T cells be pancreas infiltrating T cells or will a large number be from blood within the tissue?
No worries. Glad to hear you were able to pick up infiltrating T cells. We never used perfusion when isolating pancreatic T cells. The only time I used perfusion was when studying vascular leakage using Evans blue. It is also recommended when studying immune cells in highly vascularised organs. In my mind, pancreas doesn't seem to be one of those. Also 99% of blood is made up of RBCs and that means that before you see T cells from the blood in your organ, you would see RBCs choking it up. I would consider perfusion only if your pancreas are red instead of white. I highly doubt that is the case.
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