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Cytocentric Visionaries: Tim Bushnell

Do We Need to Think About The Cells’ Environment During Cell Sorts?


Dr. Tim Bushnell is the Director of the Research Core Facilities at the University of Rochester Medical Center in Rochester, NY. He is a globally-recognized expert in flow cytometry, serving on the Executive Committee of ISAC. He also founded the Western New York Flow User’s Group, and is co-founder of ExCyte, a company that provides master classes in flow cytometry.
Previously, Dr. Alicia Henn, Chief Scientific Officer of BioSpherix, interviewed Dr. Bushnell on LinkedIn about Key Points for Reproducibility in Flow Cytometry
Here, Dr. Henn talks with him about control of the cellular environment during cell sorts.

Alicia Henn: We think of you as a Cytocentric Visionary because of your work on trying to get flow cytometry to best detect the true state of the cell. What do you think the possibility is of getting good environmental control around flow samples as they’re being run or as they’re being prepared? I’m talking about controlling the gases and the chance of contamination, as well as the temperature.
TB: It’s not on the radar because a lot of people just stain samples on the bench and they run them in an instrument and they’re done. We had talked with some folks at Stanford from the Herzenberg Lab many years ago, and they turned us on to how we pressurize a cell sorter. We started a system where we would pressurize our instrument using nitrogen rather than room air. Our systems when we sort, are running at 40 to 70 psi so that changes the oxygen balance on the cells.
And we did a couple of preliminary experiments with a researcher here looking at the impact of that on stem cell differentiation. We were able to show that depending on how we pressurized the cells, either under normal room air oxygen to pressurize versus using the nitrogen tanks, there were differential developmental outcomes.
That really says to me that in stem cells, and probably many other cells, that we really need to think about how do we a put these cells in and get consistent cells out. And what is the impact of running cells on a cell sorter at 70 psi with a very different oxygen ratio than at room temperature or in the body.


AH: So it’s kind of a hyperbaric oxygen treatment for the cells as they’re being sorted?
TB: Yes. They’re pressurized and depending how long they sit in the tube they could be in that sample for an hour or more before they eventually get back to atmosphere.


AH: Cells can react to oxygen changes in as little as five minutes[1]. HIF proteins are regulated at the protein level rather than the RNA level. An hour under hyperbaric oxygen is a long time for cells. I can see why it had an effect on cell differentiation.
TB: Yeah that was what we thought too. That investigator went on to warmer pastures than here, but it’s something we’re capable of doing again. We have the set up. We haven’t done enough work to prove fundamentally that this is the way we should be doing things.
But I think the other side of the equation is that there are technologies with microcapillary fluidics that don’t require pressurization. In that respect, you can put one of these in an environmental chamber because they don’t require the huge pumps that the FACSAria requires. You can get a benchtop system that would fit in an environmental chamber where you could control very precisely the conditions. You could work with the cells in that environment and sort them in that condition.


Thank you, Dr. Bushnell, for your time and your insights.
For more about Flow Cytometry, see the ExCyte Blog.
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1. Jewell, U.R., et al., Induction of HIF-1alpha in response to hypoxia is instantaneous. FASEB J, 2001. 15(7): p. 1312-4.