We are subsequently staining the cells for Flow cytometric analysis and observed that the expression of some surface markers is affected by the contained enzymes. Did anyone observe the same?
Which markers are you studying? I'm also using this kit for melanoma tumors...
Hi Galaxia. We saw that CD8, CD25 and CD45RA are chopped off, at least partially. Could be that some clones will still give a decreased signal, we saw that for CD8. Are you using these markers? Do you see the same, e.g. decreased CD8 expression or low number of TEMRA/Naive cells?
I'm working with a mouse model and I don't have any problem with those markers. I'm sorry I can't help you. Have you tried any other digestion solution? Good luck
Thanks! Anyway, you should definitely be aware that the enzymes can cleave some surface markers and test this... Cheers
Hello,
I want to use this Tumor Dissociation Kit but is it mandatory to use together with the GentleMACS Dissociator. And could you please tell me your enzyme mixture for tumor dissociation?
Thanks a lot.
The KIt contains 3 enzymes (what these are is not described). The tumor tissue is incubated with the enzymes for 1 hour in total. During that time, the tissue is additionally separated by mechanical degradation using the gentleMACS. You could theoretically use another method here as well, but i have no experience with that...
Hi ,
Maybe I am coming too late but I think this kit contain Dispase and this enzyme was describe to cut some Lymphocytes receptors including CD8.. Maybe it is an explanation..
Maybe you should try with the Liberase which works really well
Good luck!
Hi Camille. Yes, we have solved the problem in the meantime :-D Dispase was not the bad guy...
At the end, no matter which enzymes you use, I can only strongly recommend to test intensively if your surface marker of interest is choped off or not!
Cheers
Do you have reproducible data using this kit? because it expires after only 3 months...
We are working with human tumors... I have isolated TILs from >100 tumors and we don't see any strong abnormalities beyond tumor heterogeneity.
Seems like the delivery time is delayed in your place, i have ordered a kit in July and it expires in Feb 2017...
I guess, it so much time past on this thread...@Henning, if no answer yet, you can use collagenase 1 plus DNAse. Otherwise you can still digest tumors with a very minimal to none dissociation cocktail. Some tumors may need modification to what recommended. Hope this helps.
Please post if you found other answers. Best wishes.
- Badges
- Science method