From: Snider Denis (sniderd@fhs.csu.McMaster.CA)
Date: Sun Jul 12 1998 - 10:14:15 EST
Hello Chris, everyone.. This response by Chris brings up a continuing issue within our facility regarding what blood preparation to use for various functional assays. I think we would all agree that heparin or EDTA are basically equivalent in regard to straightforward surface phenotyping, but functional assays on isolated cells is a different matter. We had decided recently that only heparin tubes should be used for activation and function in whole blood preparations, because EDTA acts by removing divalent cations that would be important in various signaling, enzyme, receptor or adhesion interactions. Our particular concerns are for lymphocyte, monocyte and neutrophil activation and function especially if activation is done in whole blood preparations. For instance, intracellular cyokines, surface activation markers on lymphocytes, Fc receptor function, and the diH-Rod123 assay of neutrophil function for CGD all have potential for altered function if divalent cations are chelated. I'd like to hear some response and opinion on this from the list. Denis ----------------------------------------------------------- Denis Snider Ph.D., F.C.A.C.B. Associate Professor Dept. of Pathology and Molecular Medicine McMaster University -------------------------------------------------------------------- > -----Original Message----- > From: Chris Bridts [SMTP:bridts@uia.ua.ac.be] > Sent: Thursday, July 09, 1998 4:47 AM > To: Cytometry Mailing List > Subject: Re: Clumping Cells > > Dear Avis, > > Yes, we had the same problem some years ago. > We found that sodium heparin tubes are not completely endotoxin free. > When > we looked at adhesion molecules like CD11B/CD18 they were always > upregulated in comparison with EDTA tubes or pharmaceutical heparin. > Since > then we use EDTA blood for all our functional assays, spinning the > buffy coat > cells 2x at low speed (200 g) to wash away the platelets. > > > Hopefully this helps > > chris bridts >
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