We sort platelets routinely into 96 well plates using a FACSaria. The platelets are in a standard EDTA anticoagulant bottle , same as is used for routine blood counting . The set up is to add 1ul of whole blood into 2 ml of sheath fluid , the scatter is set to log forward and side scatter and threshold is set on FS 200 , this setting can vary slightly. Initially you will see the majority of events as red cells to the upper right of the scatter plot , platelets are situated as a tail below and slightly left of this red cell population. The gate is fixed to a position so to avoid the low scatter red cell events and the low platelet events that may contain some noise and debris. If you wish you can confirm the purity of this gate by using a CD61 marker however we find that this is not necessary. The set up for our Aria is , 100 micron nozzle , pressure 20 PSI , blue laser at 100mw .we have not had problems associated with platelet clumping or aggregation within the tubing , however I can not comment on the activation of the platelets as we do not test for this , in addition the sorting and anticoagulant used will have to be appropriate to your downstream application. Hope this is helpful Ian Ian Dimmick Flow Cytometry Core Facility Manager North East England Stem Cell Institute Bioscience Centre International Centre for life Newcastle Upon Tyne NE1 3BZ UK Ian.Dimmick@ncl.ac.uk Tel 0044 191 2418831 Fax 0044 191 2418666 (mob) 0044 7970344823 -----Original Message----- From: Benoit Labarthe [mailto:benoitlabarthe@gmail.com] Sent: Fri 11-Apr-08 3:08 To: cyto-inbox Subject: Blood platelet sorting using a BD Aria... Hello Flowers! We are starting to sort blood platelets using a BD Aria. Our first experiment was relatively successful for a first short run but does anybody have experience and advices with this type of sorting? Is there an increased risk of clotting inside the tubing? Are the platelets partially or totally activated during the sorting? I guess that the pressure, laser and the electrostatic charge will not leave platelets unmodified... Since platelets may also be activated during a long sorting, we are planning to use high concentrations of platelet inhibitors but, is there a maximum length to avoid an excessive activation? Thank you all! benoit -- Benoît Labarthe, PharmD Institut de Cardiologie de Montréal Montreal Heart Institute Centre de Recherche, Laboratoire du Dr.Théroux 5000 Bélanger Est Montréal Québec Canada H1T 1C8 Tel (514)-376 3330 ext 3017 Fax (514)- 376 1076Received on Mon Apr 14 16:18:00 2008
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