FACS on platelets

Darren Hickerson dhickerson at aldagen.com
Fri Sep 28 14:27:59 EDT 2007


Greetings,
These references are from a past life, but might be helpful.  The keys are
in proper anticoagulants, buffers, centrifugation (or column separation),
temperature, and handling.  We ran live platelets daily for flow analysis
and sorting, performed live thrombin and ionophore activation during
acquisition on the flow cytometer (did a master's thesis using this
technique), and also ran tons of fixed platelets.  Arthur Bode's proprietary
method is fixing platelets and maintinaing their various properties.  Look
up more of his papers for fixing methods and uses.
Bode AP and Hickerson DHM: Characterization and quantitation by flow
cytometry of membranous microparticles formed during activation of platelet
suspensions with ionophore or thrombin. Platelets 11:259-271, 2000.
Hickerson DHM and Bode AP: Flow cytometry of platelets for clinical
analysis. Book chapter in: "Hematology / Oncology Clinics of America: Flow
Cytometry and its Applications in Hematology and Oncology" 16(2):421-54,
April 2002.
Darren Hickerson
Senior Scientist, Cell Sorting
Aldagen, Inc.
2810 Meridian Parkway Suite 148
Durham, NC 27713
(919) 484-2571, ext. 240 (desk) or 226 (lab)
Cell: (919) 599-2980, Fax: (919) 484-8792
dhickerson at aldagen.com

  _____  

From: Gary Warnes [mailto:g.warnes at qmul.ac.uk] 
Sent: Friday, September 21, 2007 3:49 AM
To: cyto-inbox
Subject: RE: FACS on platelets



Dear Sergey,

		Fixing is the last thing you do to platelets, in fact it's a
definite no-way. You mention you would like to stop clogging the machine -
if are using thrombin or ADP to activate platelets then I found a way to
stop this to use a density gradient after making PRP to purify the platelets
this also removes clotting factors and hence the platelets do not aggregate.
I guess if you wish to add thrombin to diluted whole blood then if you add
excess EDTA, removing the calcium also stops the platelets  from
aggregating. I've been out of the field for sometime but searching under
Michelson should give you lots of up to date knowledge on the subject.


Regards


Gary


Dr. Gary Warnes

Imaging & Flow Cytometry Core Facilities

Institute of Cell & Molecular Science

Queen Mary London University

4 Newark Street

London 

E1 2 AT

UK

Tel 0207-882-2402


From: Sergey Dzekunov [mailto:sergeyd at maxcyte.com] 
Sent: 20 September 2007 15:06
To: cyto-inbox
Subject: FACS on platelets


I am looking for an advice about running human platelets through a flow
cytometer.

We understand that platelets need to be fixed prior to the procedure to
prevent clogging.

Can somebody please refer me to a proper protocol?

Thank you!


Sergey M. Dzekunov



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