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I am currently trying to develop a model which measures oxidative burst and
reactive oxygen species in the HUVEC line (Clonetics). I have used DCF or DHR
to measure H2O2 production, and HE to measure superoxide anion production. My
stimulus is a mega dose of TNFa added to the cells after they have been removed
from the flask with trypsin. My problem is that I cannot detect a significant
change in fluorescence from the TNFa stimulated cells over the unstimulated
cells. My time course is usually from 15min to 120min. Previously reported
data of neutrophils indicates the ability to detect large increases in ROS
production using the same probes, the difference being a suspension cell versus
an adherent cell.
My questions are these:
1. Does anyone know anything about this cell line that would indicate it's
inability to produce significant levels of ROS?
2. Is the trypsin step cleaving the TNFa receptors which would impair the
ability to get the stimulus into the cell?
3. Has anyone had experience with the above probes? What are their
limitations and would these be possible explanations?
4. Does anyone see anything else wrong with the steps I've taken to
generate oxidative burst from cytokine stimulated HUVEC cells? What
are your suggestions? I am using an adapted method from the Handbook
of Flow Cytometry, Robinson, 1993.
5. If and when I ever generate a significant increase in fluorescence with
TNFa above the control, what is the best way to report this data. I
collect the DHR on a linear scale, and the HE on a log scale. My goal
is a screening tool to measure drug effects on reducing ROS production
I've previously been reporting any data I've generated as percent
increase above control. I use the mean channel fluorescence to
calculate this. Is this appropriate?
6. Am I trying to squeeze blood from a turnip, or should I cut the bait
and move on to better waters?
Any assistance in this matter will be greatly appreciated!
Thanks,
Jodi McKenzie-Kroeger
Procter and Gamble Company-MVL
11810 East Miami River Road
Ross, Ohio 45061
(513)627-0537
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web