Most, if not all, of your problem is likely to stem from your model.
Camptothecin is a topoisomerase I inhibitor. As such, it binds to
the enzyme-DNA complex, stabilizing it and preventing religation of
the DNA single strand break. This, by itself is necessary but
probably not sufficient to induce an apoptotic response. The
conventional wisdom is that collision of the DNA polymerase molecule
with the stabilized complex during replication causes a double strand
break which triggers apoptosis when the number of strand breaks
exceed some threshold. Simply stated, you need the cells to be going
through DNA synthesis to get camptothecin to cause apoptosis and even
then, it will only occur in S phase cells. In fact, if you inhibit
DNA synthesis in an actively growing cell line prior to addition of
camptothecin you will not see any apoptosis even at the appropriate
concentration which, by the way, is in the range of 150 nM. The small
and variable amount of apoptosis you seem to be observing is either
spontaneous apoptosis which will occur in mononuclear cell cultures
over time. If any granulocyte elements remain in your system they
will also contribute to this population. You will probably need to
trigger apoptosis through cell surface Fas receptors or by using
agents which do not require cell proliferation for activity.
Hope this helps.
Frank Traganos, Ph.D.
Cancer Research Institute at NY Medical College
>>> Ryan Hung <rhung@vcn.bc.ca> 6/9/97, 02:58pm >>>
I've just started using FACS analysis for analyzing apoptosis in PBMC
a couple of months ago. I figured that I would first try the
inexpensive technique of PI staining. However, to date, having tried
both direct staining with PI (with sodium citrate, Triton X-100,
RNAase A) and ethanol-fixation prior to PI staining, I have not been
able to get either to work reliably.
My model system itself consists of PBMC extracted from healthy human
donors at a concentration of 3x10^6 cells/ml in 1ml of RPMI growth
medium in 24 well plates. I'm using camptothecin in a concentration
of 5 ug/ml as my positive control of apoptosis. I incubate for
between 14-18h at
37oC in 5% CO2.
With direct staining with PI, I've typically obtained two
closely-spaced high fluorescent intensity peaks when reading FL3 on a
histogram plot. I see virtually no lower fluorescent intensity
peaks.
With ethanol fixation, I've had problems with gating when using a
linear scale on FSC, so I've tried using a log scale. I understand
from previous discussion that this may be incorporating significant
amounts of cellular debris. What I observe in terms of histograms
are a single high-fluorescence peak, with a lower fluorescent peak at
about 1/5 the intensity of the high peak, rather than the expected
1/2.
Since my results from both techniques don't seem to correspond well
with what I've found in literature, I've been finding it difficult to
analyze.
Any ideas or suggestions would be much appreciated.
Ryan.
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