HI

For preparation of single cell suspension from human tumor tissue we use 0.05% collagenase, 0.02% DNase and 5U/ml hyaluronidase [Sigma-Aldrich]) as final concentration with tumor tissue sample. Prepare 10X concentrated stock of the enzymes and add to the medium (10-20 ml depending upon the size of tumor tissue) with the tissue with 1X as final concentration. Incubate for 2h at 37 degree celsius with intermittent shaking. If incubator shaker is not available, use normal shaker at RT.

If you are interested in tissue infiltrating lymphocytes, do not use trypsin in enzyme mixture while preparing single cell suspension as it affects the surface expression of antigens on lymphocytes.

We have had success with many tumor tissue types using Miltenyi's tissue dissociation kit. We have not tested the affects of this kit on surface expression of antigens yet though.

@benedetta colmegna

Hi, I have the same question. Could you let me know the protocol you finally ended up using?