We are considering using a MACS machine/kit for dissociation but got low viability in our sample run and want to compare results with a manual method. I found this one:
http://www.med.umich.edu/wicha-lab/SOP/SOP%202.4-%20Mouse%20tumor%20primary%20tumor%20xenograft%20dissoc.pdf
Has anyone used this before? Any suggestions or tips to get clean single cell suspensions for flow cytometry? We are mostly interested in the infiltrating immune cells rather than cancer cells.
We used a similar protocol and is working well for single cell suspensions from mouse xenografts.
I have never used this on solid tumors, only biopsies sent for analysis suspected of hematopoietic malginancy:
Heinlein, C., et al., A rapid and optimization-free procedure allows the in vivo detection of subtle cell cycle and ploidy alterations in tissues by flow cytometry. Cell Cycle, 2010. 9(17): p. 3584-90.
Works well for me so far in a multitude of tissues sent. Some, such as a skin biopsy were a bit challenging, but worked. If I'm at all worried about viability, I check with 7AAD. After final resuspension, I stain the cells according to normal protocol. In other words, I do a full immunophenotyping, not just a DNA ploidy assay.
Hi Elizabeth,
I used a protocol very similar to this one routinely for about four years and it worked very well for us. Some of the differences with ours: (1) we only used one collagenase step with the minced tumors and digested in a sterile petri dish 1-2 hours in our 37 C incubator, using a pipet every 15 - 30 min to further break up tumor pieces; (2) after digestion and washing the cells 3x by spinning/resuspension with 20 - 30 ml HBSS to remove collagenase, we performed only 2 filtrations, first the 100 um, then the 40 um; the cell pellet after this was either used for subsequent steps or resuspended in freezing medium and placed at -80 C; (3) for our purposes, we were interested in a small subpopulation of tumor cells and so were interested in obtaining the highest viability possible; because of this, we used magnetic beads coated with an antibody directed against a surface antigen on our cells. It is very gentle and seemed to work beautifully. You might consider the same approach using an antigen specific for your immune cells of interest. Let me know if you would like further info.
you can try " the disaggregation of Hodgkin' s disease lymphnodes: a preparative technique using a new dissociation chamber" Haematologica 1987. I used it successfully to obtain a single cell suspension as a preliminary step to isolate Reed-Sternberg cells. Blood 1989.
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