We use the lympholyte M from Cedarlane with success. The volume of cells, centrifuge tube, volume of lympholyte M, number of cells per tube, cells/ml, temperature of media and lympholyte M and centrifuge speed and temperature are all critical. It has to be room temperature. It can take hours to bring your centrifuge up to room temp. We keep one or two at room temp for this reason.

The comment on smaller tumors is also important as you get a lot of necrosis/inflammatory cells with larger B16 tumors.

Its critical to use high quality collogenase as there are a lot of protease in the cheaper grades. i.e. you can strip your membrane antigens. We also add 5% FBS to the mix to further neutralize any proteases. I would focus on the use of DNAse rather than Dispase.

There are other techniques. Check out some of the older papers by Bob Wiltrout.

I have had tremendous difficulty with B16 melanoma and immune cell extraction. I have tried Ficoll (By itself and using 100%/75% gradient) and Percoll (100%/80%/60%/40%). I find these gradients don't separate the immune cells form tumors well enough, though Percoll is better than Ficoll. This may be due to the low number of T cells. I have also used CD4+/CD8+ magnetic beads but if they do work I only get back around 1 million cells when I combine a few tumors together. The best success I have had was to use a Pan T cell kit (with CD105 that binds onto the B16 cells) and that still wasn't too efficient, but it was enough for me to run FACS for analysis of my TILs.

What are you planning on doing after cell isolation? How pure do you need these samples? You could first try CD105 negative selection to remove a lot of tumor cells, then use MAC Beads for positive selection for your immune cells but that doesn't help with your cost issue.

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

I also have checked the CD105 expression on B16 and found it to be 99%+.

Oh and I should mention I have done digestion with Collagenase/DNase, Liberase, or no digestion and I found either digestion works and is better than no digestion.

The last thing to keep in mind is tumor size. The number of TILs depends on the size and age of the tumor. I have talked to a few people myself and they say stick with a smaller 10mm tumor (10-15 days) to get the best recovery.

We use the lympholyte M from Cedarlane with success. The volume of cells, centrifuge tube, volume of lympholyte M, number of cells per tube, cells/ml, temperature of media and lympholyte M and centrifuge speed and temperature are all critical. It has to be room temperature. It can take hours to bring your centrifuge up to room temp. We keep one or two at room temp for this reason.

The comment on smaller tumors is also important as you get a lot of necrosis/inflammatory cells with larger B16 tumors.

Its critical to use high quality collogenase as there are a lot of protease in the cheaper grades. i.e. you can strip your membrane antigens. We also add 5% FBS to the mix to further neutralize any proteases. I would focus on the use of DNAse rather than Dispase.

There are other techniques. Check out some of the older papers by Bob Wiltrout.

Can you tell use the volume of cells, # of cells per tube, volume of lympholyte M, size of tube, temperature of media/lymph M, centrifuge speed, accel/deccel of centrifuge, time of centrifuge, max size of tumors, and the # of cells you isolate per tube?

Can you tell use the volume of cells? Depends on the size of the tube. My preference is for a 15 ml conical bottom tube. However, if you are doing a lot of cells use a 50 ml tube. You can do this with up to but no more than 2x10e7 cells/ml. Better to do with 1x10e7/ml. I would have about 4-5 mls of lympholyte-M on the bottom and about 7-8 mls of cell suspension layered on top.

# of cells per tube, No more than 2x10e8 cells per 15 ml tube.

volume of lympholyte M, 5 mls would be best. But you can use a little less. Work out what works for you. Be very careful when layering the cells over the lympholyte M. They will start separating within a short period of time.

size of tube, 15 ml or 50 ml. Best is 15 ml conical.

temperature of media/lymph M, Must be room temp (about 22C)! Also the centrifuge should be at room temp.

centrifuge speed, about 1400 xG. This is not rpm! Calculate the rpm for each centrifuge you are using. The optimal xG will be lab dependent. Somewhere between 1250 and 1500 xG.

accel/deccel of centrifuge, No break no fast acceleration.

time of centrifuge, 20 min

max size of tumors, about 1 cm or whatever does not result in a lot of necrosis. B16 is really friable.

and the # of cells you isolate per tube? Highly tumor dependent. Also depends on the size of the tumor. Note, I have over 50 variants of B16 and each has a different NPC infiltrate characteristic. Most common are F1, F10 and BL-6. If you check our old papers on doing this with B16 you should find the answer for these three variants. We have been using mammary tumors for the last decade and so don’t remember.

Macrophage frequency in B16. Note long before we knew what MDSCs were.

Talmadge, JAMES E, MARC Key, and ISAIAH J Fidler. “Macrophage content of metastatic and nonmetastatic rodent neoplasms.” The Journal of Immunology. Am Assoc Immnol, 1981.

Key, M, J E Talmadge, and I J Fidler. “Lack of correlation between the progressive growth of spontaneous metastases and their content of infiltrating macrophages.” Journal of the Reticuloendothelial Society, 1982.

How to isolate NPC from tumors, lots more if you do a pub med search

Younos, I. H., A. J. Dafferner, D. Gulen, H. C. Britton, and J. E. Talmadge. “Tumor regulation of myeloid-derived suppressor cell proliferation and trafficking.” International immunopharmacology. Elsevier, 2012.

Cancer

http://link.springer.com/article/10.1134/S0022093013020175

Dear Dr. Talmadge,

Thank you very much for such detailed explanation. My last test was done with 2 step isolation. First I minced the tumor (the diameter was approximately 12mm) and treated with collagenase (Sigma) and dispase (Roche). The enzymatic reaction was stopped by addition of Na2EDTA. Then the lysate was filtered through 100um and then 40um filters and washed.

Pellet was completely black. I resuspended it in 1x PBS and overlayed on Histopaque 1083 (SigmaAldrich). I used 15ml conical tube and spun the sample at RT, 450g, 25 min. Although I still got the black debris in the interphase.

I can try your protocol with Lymphocyte M and hopefully it will work in my hands too.

You can expect some black in the interface as the macs in the interface will have phagocytized some tumor cells and look black. Confirm what you have by flow and cytospin.

If you are concerned about time, you can try the Easy-Sep from Stem Cell Technologies, as it requires only 5 minutes to allow beads to attach to tube wall and there is no need to wash with several volumes of buffer. You can use the same CD45 beads you have for MACS.

I think 12mm is going to hurt you.

I just came back from an experiment where I had 13mm tumors on B6 mice, I minced them, digested with Collagenase/DNase. Removed red blood cells with ACK Buffer and used the Pan T Cell Kit from Miltenyi Biotec to negatively select T cells. I have tested the column with lymph nodes and spleen and get >90% purity. But with the 13mm tumor, after digestion, ack and the MAC beads I only had 7000 cells within a total of 8.5 million cells that were ran through the machine (0.08%). I looked at the column flow through cells (which are the t cells since it is a negative selection kit) and the bound cells (The non T cells) and found that the bound cells had less than 50 t cells in a million (0.005%). So the column worked well, I literally just had 7000 t cells in 8.5 million.

My guess is that the tumor size was too large and my immune cells were dieing out. Mind you I used a Pan T cell kit and analyzed flow cytometry with CD4/CD8 which may explain why I get less purity than a positive MACS bead kit using CD45. But as I said before, I find MAC beads work a lot better than Ficoll and Percoll. Though I have been using large tumors >13mm diameter, and starting next week will try with tumors around 0.5-1.0mm.

You should compare Dispase to DNase, because if your digestion doesn't work well then your immune cells will be pulled down with your tumor cells in the histopaque column.

Hope this helps!