Hello to everybody,
I want to isolate all the leukocyte populations (CD45) from B16F10 tumors (injected in mice subcutaneously). Can someone recommend a good dissociation media (like PBS or complete RPMI)? In particular, which enzymes would you use? I know that some of them can degrade surface antigens.
Thank you very much,
Michele
Hi Michele,
I strongly suggest you to use the magnetic bead–based separation, which offers easy handling and reliable results. All the main biotech company sell these beads, and the prize are affordable. Smash the tumour mass onto a 0.70 um cell strainer, and filter again the cell suspension through a 0.45 um sterile filter. I recommend you to use RPMI, with high and stable glutamine, without serum added (better than PBS, however without Ca2+ and Mg2+ to decrease cell attachment during the purification step). By this way, it is possible to limit leukocyte aggregation and activation (especially for monocytes). Exactly, there is no need to use an enzymatic approach to digest the tumor mass: for sure, you damage surface antigens. We are working close, we can enjoy an Italian espresso and discuss more about the experimental details. Let's meet!
Damiano
Suggest that you look into the use of collagenase / DNAse digestion. Smashing through a cell strainer kills a lot of cells and you loose frequency data. Also the released DNA can aggregate cells. We and many others have published the digestion approach. Beads are of course one approach. Alternatively, you can largely separate parenchymal cells from leukocytes by density gradient centrifugation. Again see various papers on techniques. You might then use beads for a final separation of specification cell types.
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