In answer to Jodi L. McKenzie-Kroeger's question:
Subtracting the baseline of the control cells from the stimulated
cells is the correct way to do this assay. No matter how well you do
the assay, or prepare thecells there will be some baseline change.
It is not very much with DHR123 - and that one of the reasons it's a
good probe, but there will probably be some leakage and you need to
take this into account. With really leaky probes like DCF - you have
no choice but subtract baseline or your results are trash!
Paul Robinson
I have a basic data analysis question. I am measuring oxidative burst from PMNs
and Monos using DHR123. I am stimulating my cells with TNFa and treating them
with actives to inhibit ROS. My controls include DHR only cells and DHR/TNFa
cells. I have previously been comparing my compound treated cells to the
DHR/TNFa cells without subtracting out the baseline DHR stained cells. This is
how I've seen it reported in the literature, and I've previously been advised
not to do so. However, I have now been asked to subtract out the baseline DHR
stained fluorescence to compare the treated vs. untreated cells. I am not
comfortable doing this, but I'm not sure why I'm uncomfortable. So, here are my
questions:
1) Is it okay to subtract out the baseline fluorescence value from the
stimulated cells in order to analyze the data?
2) If not, please give me a good explanation to pass on to my supervisor
(non-flowist) so that I can get over this particular hurdle.
3) If so, please give me a good explanation so that I can deal with my
discomfort.
Thank you in advance....Jodi L. McKenzie-Kroeger
J.Paul Robinson, Purdue University Cytometry Labs
Professor of Immunopharmacology
robinson@flowcyt.cyto.purdue.edu PH:765-494 6449 FAX:765-494 0517
web http://www.cyto.purdue.edu
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