Summary Document
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Hello all,
Here are all the responses on platelet dust. I want to thank everyone
who responded.
Bhargavi Rajan
Technical Coordinator, Flow Cytometry Facility
Children's Research Institute
Children's National Medical Center
111 Michigan Ave, Washington DC-20010
PHONE: 202 884 2308
FAX: 202 884 3929
EMAIL: brajan@cnmc.org
Question:
I have an investigator who wants to track activated platelets-these are
about 1-3uM in size. Apart from these she also wants to surface
expression of CD42b on "platelet dust"; these are about 0.02-0.05uM in
size.
Do you think the FACSCalibur is able to distinguish between platelet
"dust" and say noise? Do I have to set the instrument with beads that
are that size? Do you know if such beads are available?
Responses:
1. Cellular "dust" is released from many cell types under stress or
activation. As the particles are below resolution for size, it is common
to resolve them from instrument noise by triggering (thresholding) on
fluorescence as opposed to scatter. In this case you could label
platelet dust preparations with fluor-tagged
CD62, as the triggering signal, and CD42b conjugated to another fluor,
effectively identifying only platelet derived particles and their
expression of 42b. You will find many articles in journals specializing
in hemostasis ie Blood which use this technique. Some have gone to
equipping analysers with photomultipliers for scatter signals but you
will find the fl threshold technique works well on a calibur.
I hope this helps.
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2. Many years ago I worked with an investigator that studied Platelet
Dust. He published quite a few papers on it. Art Bode. The Calibur is
capable of resolving the Dust. We triggered on side scatter on a
FACScan and looked at FSC in log, I think. We used .1 uM beads as a
standard. It has been a long time, so I do not remember all the details,
but they should be in the papers.
Good luck
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3. I've had lots of experience doing just that. The Calibur is capable
of seeing the microvesicles or "dust" but you have to make sure of
many things:
a. Filter any buffers twice through 0.2um. Run diluent buffer alone
for same duration as platelet samples to determine the degree of dirt
around. If this appears high, inspect machine buffer alone, and/or
refilter diluent buffer.
b. Determine exactly where on your platelet window the microvesicles
will be found. Do this by sedimenting platelets very gently. Then
hardspin (10000RPM)to be sure whole platelets and lysed membranes are
gone. Then sediment microvesicles at 100,000xg - 1 hour (check in
literature for conditions), resuspend in small amount of liquid and run
these as well as unsedimented microvesicle super. Make sure platelet
profile is well to the right of window but completely visible.
c. Finally, use platelet protein marker to make sure you are looking at
platelet microparticles and not microvesicles of other cellular origins.
Visualizing GP42b would not distinguish between platelets and
endothelial cells, whereas 2b3a would. So use antibody to the integrin.
Should work. It has for me.
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4. We use CD42b to look at microparticles. Spherotech makes a size
calibration bead (BCP 10-5). We set the gate at all less than the lower
limit of the beads (1.09u). Below that I don't know of anyone who has
anything smaller. Spherotech might be able to make something for you.
She should look at the papers by Jimenez. Hope this helps
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5. tough one...
a. beads aren't going to work all that well as they'll most likely have
different refractive indices that the platelet microparticles...
b. have your researcher fractionate the samples so you'll have whole
blood, platelets rich serum, and finally microparticle rich fraction and
see if you can tell the difference (the last two will overlap somewhat).
As you are pushing your thresholding down, through in filtered PBS to
ensure you are not pickingup noise...
c. the Caliburs are kind of poor for this as you'll have to be in log
setting for scatter and the log amps are awefully noisey at that end of
the scale....the digital systems (Coulter or BD) will give you better
results
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6. Spherotech sells fluorescent beads with a 0.04-0.06um range.
