I normally used dynal beads and they worked fine.

but miltinyi should also have them.

It seems like the Pan T cell Isolation Kit II is what you are looking for, as it should enrich for T cells without activating them.

You could always use the CD3e beads to isolate T cells, you would just have to flush the cells off the column and basically trash anything that flows through. Not sure how the CD3 antibody will effect the activation state of the T cells so the negative selection approach from above is probably the best approach.

Personally, I prefer the easySep system to MACS. This is also a magnetic separation system but has no need of columns! I tested both of them and had much higher output rates when using EasySep.

Often you also can choose between pos. or neg. selection, whatever is more feasible for the further work with your cells.

I would think the Pan T cell kit would be your best bet. It is for "untouched" isolation of T cells, so it will basically deplete all the other cell types. Since it never touches your T cells, you won't risk activating them. It also depends on what you want to do afterwards. Recently I've been using the FlowComp magnetic beads from Invitrogen, but it's just been to isolate CD8 T cells for RNA.

What frequency of tumor-infiltrating lymphocytes is represented by T cells in your model? If it's reasonably high, as has been the case in some other tumor models I've worked with, I would suggest skipping the MACS T cell isolation step entirely since you're going to be sorting the cells them by FACS anyway.

You're inevitably going to get some cell loss in a magnetic enrichment step, and I find that this is sometimes worse when trying to negatively isolate highly activated T cells like those from tumors (I suspect that it's because they express a broader range of integrins and activation markers than naive T cells and consequently adhere to the columns or other cells in the columns).

In my experience, I've gotten better cell yields by taking my Percoll-enriched lymphocyte fraction and just staining it directly for FACS.

That being said, when working with very rare cells I tend to do a MACS pre-enrichment if only to reduce my time on the sorter to a reasonable amount.

Use Pan T cell kit would be better to meet your need. To get a higher purity due to it's a negative selection, you may use LD column from Miltenyi.

Just realized that since I am taking a tumor digest to isolate TILs, The tumor cells may not be labelled with the Pan T cell kit and will be depleted with the T cells. So I decided to look into what cells this kit depletes.

Here is what the kit isolated:

*CD11b - Dendritic Cells

*CD11c - Dendritic Cells

*CD19 - B Cells

*CD45R (B220) - B Cells

*CD49b (DX5) - NK Cells

*CD105 - tumor-associated angiogenic vascular endothelium

*Anti-MHC-class II - dendritic cells, monocytes/macrophages, B cells in lymphoid and non-lymphoid tissue, thymic epithelial cells, and on subsets of hematopoietic progenitor cells in the bone marrow.

*Ter-119 - Erythroid cells

After finding the above info I was scared B16F10 wouldn't be depleted so I looked into MHC Class II and CD105:

I found a paper that says: B16F10 is a spontaneously arising melanoma cell line derived from the C57BL/6 mouse that shows very low expression of MHC class I and no expression of MHC class II. (Fidler I. J. Biological behavior of malignant melanoma cells correlated to their survival in vivo. Cancer Res., 35: 218-234, 1975.)

Another paper I found shows that B16F10 highly expresses CD105. (Jarosz M et al. Therapeutic antitumor potential of endoglin-based DNA vaccine combined with immunomodulatory agents. Gene Ther., 20: 262-273, 2013.)

http://www.nature.com/gt/journal/v20/n3/fig_tab/gt201228f1.html#figure-title

Therefore it is hopeful that the Pan T Cell Kit can isolated untouched T cells from a digestion of B16F10 melanoma. Using this kit with the MAC Beads magnetic and LD columns should theoretically work!

Hi Nathan!

I am with STEMCELL Technologies, and I cover the Ontario region. I noticed that Sophie Kitzmuller recommended you our EasySep system over the Miltenyi one. As she mentioned, you can do a total T cell enrichment without columns and get untouched cells back. In this particular case, the protocol is quite convenient as it takes only 15 min.

I will say that Adeeb asked a great question regarding the frequency of T cells in your tissue. Both Miltenyi's and our kits are optimized for spleen samples, and the degree of enrichment on other tissue types will depend on the relative frequency.

If you are interested in an alternative method, you can see the kit at: http://www.stemcell.com/en/Products/All-Products/EasySep-Mouse-T-Cell-Isolation-Kit.aspx

If you want more info, email me at [email protected]

Im a little late to this conversation, but has anyone ever quantified the total number of CD4 and CD8 T cells that infiltrate into a B16-F10 tumor?

Best,

Thomas