Michael, with regard to your problem with ionophore and platelet activation. In my experience A23187 can be very labile under certain conditions. Here are some of the standard methods I use to ensure consistent activation. I use the free acid form from Calbiochem (supplied by CN biosciences cat:100105). Make up a stock 1mg/ml solution in DMSO Store in 50 microlitre aliquots at -80. I have stored at -20 at the bottom of a chest freezer. For use remove from the -80 and leave to thaw at room temperature in the dark. DO NOT warm in the hand or waterbath. For around concentration 2x10^9/L you need about 5-10 microlitres/100 microlites of platelets of a 1 in 10-20 dilution depending on the effect you are looking for (MIX WELL). These values will give you significant activation and consistent binding of FITC annexin V (>90% positive events). Incubation times vary with final concentration, 5-15 min with those described above. However, I suggest you optimize for your system. I wouldn't recommend a wash step, because unless you have really well washed platelets or GPIIbIIIa blocker the platelets will clump in a mass at the bottom of your tube. I keep the unused stock in the fridge for about a week and the -80 stock for about 2 months as per data sheet. As an alternative, have you thought about using thrombin or TRAP to induce P-selectin expression? Both of these are used regularly in the literature (and in Purdue archives), and I have used both for positive controls of P-selectin expression. Hope all this helps Craig Turner CDL NBS UK ---------- From: MSuter@omlabs.com To: cyto-inbox Subject: Platelet activation studies Date: Thursday, January 24, 2002 1:48AM <<File Attachment: ALTERNATIVE.HTM>> Greetings: I am working up a flow cytometry method for heparin induced thrombocytopenia (HIT), using incubation of normal platelets and heparin in the presence of patient plasma, followed by measurement of platelet activation using CD62p. This is following the method of Tomer (Journal of Hematology, 1997, 98, 648-656). The source article describes production of a positive control by incubation of platelets with calcium ionophore A23817. I am having some difficulty getting this to work, and details in the article are scanty. Does anyone have experience with use of calcium ionophore for this purpose? I'm particularly interested in: - Which salt of CaI is best (a Sigma number would be ideal)? - What is your process for preparation of the CaI (actual recipe)? - How long are platelets incubated with CaI, and are they washed prior to labeling with CD62p? - What are the stability and optimal storage conditions for CaI in solution? Thanks very much for your assistance! Michael Suter, MT Flow Cytometry Tech Specialist Oregon Medical Laboratories msuter@omlabs.com +++++++++++++++++++++++++++++++++++++++++++++++++++++++ The National Blood Service. Do something amazing today - Give Blood. Please call 0845 7 711 711. You can visit us at www.blood.co.uk, or on BBC2, Ceefax page 465. +++++++++++++++++++++++++++++++++++++++++++++++++++++++ The views expressed in this e-mail are those of the sender, and not necessarily those of the National Blood Service. This text confirms that this e-mail message and its attachments have been swept for the presence of computer viruses by the National Blood Service, however we cannot guarantee that they are virus free. All e-mails and their attachments to and from the nbs.nhs.uk domain are archived, and their contents may be monitored. +++++++++++++++++++++++++++++++++++++++++++++++++++++++
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