Re: How to prevent "sticky" cells clogging machine
As our standard treatment, the HIMC adds Benzonase to our cell media (RPMI + pen/strep/glutamine + heat-inactivated FBS) during thawing. We then do an extra benzonase-media wash after the first centrifugation; both benzonase+media washes are to help remove DNA present from cells lysed during freeze-thaw. After that, the cells in my experiments are almost always in CyFACS (PBS + 0.1% BSA + 2mM EDTA).
See here for our standard live cell surface phenotyping protocol: viewtopic.php?f=11&t=258
In my experience, yes, samples with lower viability are more likely to clump, mostly in the CyFACS *after* the 2 benzonase+media washes. So, EDTA doesn't always take care of the problem.
However, I will also state: clumping can also be a donor-specific occurrence. For example, I was recently working on a study with multiple PBMC timepoints (~6 week period) for each donor. For one donor, 0 of the 7 timepoints clumped. For another donor, 6 of the 7 timepoints clumped as soon as the cells "saw" CyFACS (ie, were taken out of benzonase+media, and before they "saw" antibodies), and continued clumping through the entire experiment. There was no significant difference in viability or recovery (>85% viability, >70% recovery) between the two donors, and the likelihood is very small that only one donor would have been mis-processed or mis-stored multiple times over the course of ~6 weeks. The time series for each (healthy) donor was the same as well, so it's unlikely that it was a reaction to the study's vaccination (also, one of the affected donor's clumping timepoints was Day 0 pre-vaccination).
So, unfortunately, sometimes these things just happen, and we haven't figured out a way to *stop* or *reverse* clumping once it starts.
Mike