> -----Original Message----- > From: Silke Schmidt [mailto:sschmidt@ukaachen.de] > Sent: Tuesday, January 08, 2002 4:18 AM > To: Cytometry Mailing List > Subject: neutrophil apoptosis at biomaterial contact > > I am testing neutrophil apoptosis in human whole blood after > contact with > various polymers used at biomaterials. > There is a group of polymers that cause high apoptosis rates > in TUNEL assay > but low rates in AnnexinV assay and also very low necrosis > rates. These are > the polymers that I know to be quite inert. > > On the other hand there are some polymers that strongly > activate neutrophils > (for example their receptor expression and IL-8 production). > They show low > apoptosis rates in TUNEL assay, high apoptosis rates in > Annexin assay and > low necrosis rates. > > These results are reproducible with a very low standard deviaton... > But I simply cannot explain them! Hi Silke, I would recommend the following additions/modifications to your experimental design: 1] include the use of a negative control and see how each assay performs. This might be neutrophils immediately upon isolation or a cell line. Consider any steps in your isolation procedure which may be inducing apoptosis or inducint artifacts in either of the assays. Does cellular adherence differ for the different biomaterials? 2] include the use of a positive control. Find a stimulus known to induce apoptosis in these cells and/or use a cell line. Not sure what this is for neutrophils as we don't work with them, but this info is probably available in the literature. 3] Examine the cells by fluorescent or electron microscopy for morphological evidecne of aopoptosis. 4] Sort the negative and positive annexin and TUNEL cells and examine them under the microscope or run a gel and look for DNA laddering. Hopefully these types of approaches will resolve the apparent discrepancy between the 2 assays. Good luck. Tom **************************************************************************** * Thomas W. Mc Closkey, Ph. D. Director of Flow Cytometry, North Shore University Hospital Assistant Professor of Pediatrics, New York University School of Medicine Boas Marks Biomedical Research Center, 350 Community Drive Manhasset, Long Island, New York 11030 ph: 516-562-4844 [office], 516-562-1135/4641 [lab] fax: 516-562-2866 **************************************************************************** *
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