Cool Flow Experiments we ran at USF

This page of the website that I am most looking forward to seeing grow.  We are so new that the lab is not even officially open, so for now this page is a bit barren.  My plan is to outline here experiments that we ran (without giving away data that might lead to the investigator getting scooped), so to illuminate various opportunities that Flow Cytometry provides, as well as potential pitfalls of exciting experiments.

May 2008

There are a bunch of fun experiments around.  One set is our effort to sort cancer progenitor cells.  Stem cells express drug pumps in order to protect them from environmental polutants.  They also pump out dyes and we are taking advantage of this: load cells with Hoechst33342 or Invitrogen DCV, then identify dim cells that exclude these dyes.  The trick is that these dyes (particularly Hoechst) is really well excited by UV lasers, and all we have is Violet.  Alas, we managed to figure out how to do this.

Also, a group intereted in Treg cells now has the USF record for the most colors in one experiment, 8.  Congrats Maura!

2007

So, here is one from Lory Lutz of the Morgan lab.  She was looking at retention of monocytes that were injected into a brain a day later.

Bone marrow cells from GFP mouse was injected, than a day later the brain was dissociated using a Miltenyi kit.  The two questions for this pilot experiments were:  Would the cells be in any decent shape after the dissociation and can we detect the injected cells?

The answer for the first question is a resounding yes: of the control (non injected) brain cells 4.3% was dead as judged by PI staining, the injected sample contained 3.7% dead cells.

The second question is tougher:  First of all, running only bone marrow cells from the GFP mouse only 18% of the cells were truly green.  Well, we would expect heterogeneity in terms of GFP expression in different cell types but this is lot less than we would hope.  So, what we found was 21 GFP positive cells out of 10, 000 events in versus 3 from the control.

Conclusion:  While the brain dissociation method seem to work well, but next time we need to make sure that the monocytes population that we are hoping to detect is actually green.  Also, being a rare event, lot higher number of events should have been analyzed.  21 out of 10, 000 extrapolate to approximately a few thousand from the full sample and analyzing substantially larger sample would be very useful.

GFP histograms from the Home of the original green mouse

GFP in green mouse bone marrow cell isolate