From: Darren Hickerson (DHICKERSON@brody.med.ecu.edu)
Date: Fri Feb 13 1998 - 13:57:14 EST
2 Things: (1) Anne: >>5ul aggregation inhibitor if desired (GPRP or RDG) What final concentration do you use of these inhibitors? (2) My turn at the platelet prep. Spin NaCIT WB (I'm also anti-EDTA for platelet work) about 500xg for about 5 min for thick, swirly PRP (can use 5ml WB or less if needed). Dilute PRP 1:100, add Ab to 0.5 - 1 ml samples, inc. 20-30 min, run without washing. If washing is needed for some other purpose, pretreat PRP for at least 5 min w/ 1 ug/ml (final) PGE-1, and complete prep work w/in 30 min. With PGE-1 tx, you can wash PRP and 1:100 diluted PLTs Ab. For no-wash prep above, take the time to titer the Abs to ensure saturation w/o gross excess (shoot for conc. giving best signal to noise ratio.) Usually only required once for each clone or lot. Thanks, 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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