I observed the decreased viability of cells after MACS sorting which influences the outcome of further experiments – 30-50% content of dead cells. Dead cells are observed both in positive and negative fraction; before sorting I do not observed decreased viability – I suspect that something happens during incubation with beads or during the separation. I follow the protocol provided by producer of beads – together with added volume of beads, time and temperature of incubation. Only exception: separation is conducted on RT, with MACS buffer with RT – when I used cooled buffers, mortality was even higher. How can I increase the viability? What viability values do you observe in your experiments?
Technical details: I work with AutoMACS Pro Separator, positive selection, beads: anti-SSEA-4, cells: Mesenchymal Stem Cells.
I have never used an AutoMACS Pro Separator, so I cannot address any technical issues related to that, but I have decades of experience using MACS separations - a lot of CD34/CD133 isolations of human cord blood and bone marrow, as well as years working on lymphoma cell lines. I always do my separations at RT. The buffers are chilled, but sit in the hood during separation, so as the isolation proceeds, the buffers are warming up.
With the CD34 and CD133 isolations, I virtually never had problems with viability. The few exceptions were samples that were mishandled. However, the lymphoma cell line isolations were much more problematic. I would go from 95%+ viable pre-isolation to ~12% or less viable post-selection in the selected fraction.
The issue turned out to occur during the labeling step, when the cells are at extremely high concentration [~2 x 10^8 per mL]. I found that with these particular cell lines, incubation of cells at that density - whether or not beads or antibodies were present - led to drastically reduced viability. I was able to figure out that this was due to Fas:FasL interactions. The cell lines I was using expressed very high levels of FasL, and being lymphocytes, also induce Fas expression at high density, so the cells were committing fratricide.
I doubt that FasL is the actor in your system, but it never hurts to check. A key point it that FasL is readily lost if you do not inhibit metalloproteinases, so the cells must be incubated with a lot of EDTA - 10 mM comes to mind - during staining and analysis.
One other thing I have found helpful is to use Miltenyi's Tyto running buffer for all steps [antibody and bead labelling, column separation, flow cytometry analysis] to prevent losses due to clumping. It is an expensive reagent, but it can save precious samples.
Gary Lee Gilmore Thank you for the suggestions! I suspected earlier that density may affect the vaibility, howerver I do not have the opportunity to try out the FASL interactions. When it comes to buffer for beads incubation, I used PBS + 2mM EDTA, what was suggested in the producer protocol.
Fas-L is extremely sensitive to cleavage by metalloproteinases such as mmp-9 and requires 10 mM EDTA to prevent loss of expression. There was a small molecule inhibitor of mmp-9 available in the past, but it was taken off market in hopes of therapeutic use. As far as I know, that has not happened, but the inhibitor is still not available for research use.