Summary Document
This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.
http://www.cyto.purdue.edu/hmarchiv/index.htm
Thank you for the answers to my query about measurement of
microparticle by flow cytometer. All the answers are grouped into 2 and
attached here. Please also refer to the old email summarized by Rajan
Bhargavi about platelet dust. It also has many helpful responses about
platelet miroparticles.
Xiaoping Wu, Ph.D.
Flow Cytometry Facility
Research Division of the Puget Sound Blood Center
My query:
I have a user who wants to study microparticles, assumably at size
0.1-3 micron. Do anyone know any commercial available microbeads at
this size? What is the hallmark surface marker for microparticles,
generated by platelets or monocytes? Any input about the study of
microparticle will be appreciated.
Response group 1:
Molecular Probes/Invitrogen has various sizes including very small
ones. -Elizabeth R. Simons, Ph.D.
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I have used polystyrene microbeads from Polysciences. These are
relatively uniform and cover the size range you requested. -Charles
Dowding
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Try Sperotech for the microparticles. Jane Miller, Dept. Micro. Mole.
Path., Texas A&M Hlth. Sci. Ctr., College Station, TX 77843 - Jane S.
Miller
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Spherotech does offer a wide range of fluorescent and non-fluorescent
particles in this size range. Please refer to
http://www.spherotech.com/ for more information or please contact me if
you have any questions. -Brian Kildew-Shah
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I recently got my 0.049uM and 0.49uM beads from Spherotech.
Spherotech has the following particles that will be fluorescent in the
FITC channel:
| FP-00552-2 | Fluorescent Yellow Particles | 1% w/v | 0.049 µm | 2 mL | $100.00 |
| FP-0252-2 | Fluorescent Yellow Particles | 1% w/v | 0.24 µm | 2 mL | $100.00 |
| FP-0552-2 | Fluorescent Yellow Particles | 1% w/v | 0.49 µm | 2 mL | $100.00 |
| CFP-0852-2 | Carboxyl Fluorescent Yellow Particles | 1% w/v | 0.85 µm | 2 mL | $115.00 |
Spherotech has the following particles that will be fluorescent with
the 635nm line:
| FP-00570-2 | Fluorescent Sky Blue Particles | 0.25% w/v | 0.09 µm | 2 mL | $100.00 |
| FP-0270-2 | Fluorescent Sky Blue Particles | 0.25 w/v | 0.22 µm | 2 mL | $100.00 |
| FP-0570-2 | Fluorescent Sky Blue Particles | 1% w/v | 0.45 µm | 2 mL | $100.00 |
| FP-0870-2 | Fluorescent Sky Blue Particles | 1% w/v | 0.84 µm | 2 mL | $105.00 |
http://www.spherotech.com/new%20downloadable%20notes/
fluorescent%20particles.pdf will provide you with additional
information regarding Spherotech fluorescent particles. -Bhargavi Rajan
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Duke scientific is also another company specializing in beads -Scott
Tighe
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for beads I can give you the folowing link, they have different sizes
and qualities.
http://208.106.133.230/www.dukescientific.com/pages/pagef316.html?s=979&ss=983&t=991
-Schade Grit
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I am not sure this is what you are looking for, but we make fluorescent
nanoparticles in that size range and smaller. We supply these
nanoparticles with a COOH-functional surface.
http://www.crystalplex.com/shop/scripts/prodList.asp?idCategory=1232.
There is a relatively straightforward conjugation procedure for
covalently attaching proteins to the surface of these nanoparticles.
You can make fluorescent probes for specific cell surface markers. I
have attached this procedure. I am not sure if there is a hallmark
surface marker for nanoparticles. -Matt Bootman
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Try Bangs Labs
http://www.bangslabs.com/index_static.php -Martin S. Zand, MD, PhD
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Interfactial Dynamics Corporation (IDC) also specializes in
microspheres. The website is http://www.idclatex.com/ -Steve Crouse
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Response group 2:
You might be interested in the 2007 regional meeting. John Nolan, for
one, will be discussing microparticles in cytometry -Allan Kachelmeier
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We use Spherotech beads # BCP-10-5 they have a lower size limit of 1.09
u and we use this to set the boundary between platelets and
microparticles. We use 42b+/31+ for PMP, 42b-/31+ or 62E+ for EMP, we
make a stab at leukocyte microparticles with 45+ but don't try to
discriminate origin. Hope this helps. -Judith Stewart
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You can buy microbeads from polysciences. They have all kinds of beads.
Dr. Michael Sefton did a lot of research on micro-paritcles, and I
believe you can check his papers for more information. -Zhengyu Pang
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John Nolan at the La Jolla Bioengineering Institute has done some work
on microparticle analysis and may be able to give you some pointers.
-David Basiji
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The published literature showing EM of microparticles shows that the
vast majority are between 100 and 200 nm in diameter. Polysciences has
a good selection of small polystyrene microspheres. Be aware however
that the refractive index of polystyrene is much higher than a lipid
vesicle, therefore they will scatter much more light. This is a
corollary of the well-known rule that: Flow cytometry light scatter
does not measure particle size. You will not be able to detect single
microparticles on the basis of light scatter. The many papers that
claim to do this are likely measuring the scatter from many coincident
microparticles.
The more appropriate approach would be to use an abundant surface
marker such as CD41, labeled with a bright dye like PE. Triggering on
fluorescence, it is just possible that you could detect an individual
microparticle, but you still must be very careful about coincidence.
The simplest way to check whether you are measuring single particles or
coincident particles is to do a series of two-fold dilutions and show
that the count (or count rate) goes down, but that the mean
fluorescence does not. If the mean fluorescence goes down as well, this
suggests that you are measuring coincident particles. -John Nolan
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