Hi,
we are preparing single cell suspensions from breast tumors for mass/flow cytometry analysis.
We have tried different dissociation protocols (collagenase I, dispase II, etc) but none of them work efficiently (low cell yield and viability). Recently we are trying Tumor Diss. Kit from Miltenyi Biotech, but again we are struggling with low cell yield (e.g. 0.5x10e6 cells per 1g tumor) and presence of viscous "mucus" in dissociation mixture. Does anyone have experience with this kit? What are your yields? Do you have any tips how to increase cell yield and protect cells during dissociation?
thanks a lot!
Barbora
Hi Barbora,
I'm keen to hear more about your progress with this method. I'm also working with the Miltenyi kits and tend to get low yields. Have you had any luck optimising dissociation? The "viscosity" could be due to presence of DNA after dissociation, which you could treat with DNA to aid culture or other downstream applications.
Hi Carlos,
Thank you. We optimized protocol and the best approach is overnight dissociation with gentle shaking using collagenase, dispase and ROCK inhibitor. With addition of DNAse in the end. We get very nice yield and viabilty. Hope you had also luck with your samples.
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