letter to cytometry mailing list: People wrote on: (1) Contradictory results (decrease in PAC-1 when it should have increased) (2) specificity of PAC-1 (3) Possible problem with EDTA as anticoagulant (4) General fluctuation in platelet Ab expression Here's our experience: (1) We have gotten less-than-spectacular or even contradictory results with PAC-1 as well, even in washed platelets (fibrinogen free). Study of literature & product insert revealed PAC-1 only expressing under some very specific activating conditions. Please review those sources to ensure PAC-1 will be a useful measure of activation in your system. It is also important to note that in a "simulated cardiopulmonary bypass," any platelets that activate may be adhering to internal surfaces of your apparatus and therefore will be out of circulation, especially if any biological material is used in the test system (shows an apparent DECREASE in activation since only monitoring suspension cells). (2) PAC-1 is specific for an activated conformational form of GPIIbIIIa (CD41), not CD62 as someone recently reported (CD62 is, however, another good activation-dependent neoantigen to monitor). (3&4) We have found that certain calcium binding compounds render the resting form of GPIIbIIIa virtually undetectable by some MoAbs, while other GP expression goes unaltered (eg., Ib) (the IIbIIIa expression increases when the coumpound is removed). Since calcium is important in forming the IIb and IIIa complex, a chelator such as EDTA may dissociate the proteins on the membrane if present at time of analysis. This may effect PAC-1 expression. Generally, though, platelets anticoagulated in EDTA and then diluted are well positive for GPIIbIIIa. I hope some of this is helpful. Darren Hickerson dhickerson@brody.med.ecu.edu Core Flow Cytometry Facility, Brody 4W37 Department of Microbiology and Immunology East Carolina University School of Medicine Greenville, NC 27858 Phone: (919) 816-2799 Fax: (919) 816-5018
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