Journal
JOURNAL OF IMMUNOLOGICAL METHODS
Volume 299, Issue 1-2, Pages 117-127Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jim.2005.02.003
Keywords
perform; flow cytometry; calcium; granzyme B; heparan sulfate proteoglycans
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How perform (PEN) delivers the granzymes during cytotoxic granule mediated apoptosis remains a mystery. A major obstacle has been the inability to visualize PEN in either monomeric or polymeric form after interaction with the target cell surface. An antibody based technique is described which detects cell surface PFN on intact cells by flow cytometry. The methodology requires the presence of calcium (Ca2+) at a concentration which supports binding but not polymerization of PFN. Functionality was ensured by showing the cell surface PFN was able to deliver GrB causing caspase-3 activation and mitochondrial depolarization. The technique demonstrates a role for heparan sulfate proteoglycans in PFN binding. Further, the variable sensitivity of effector versus target cell lines to the permeabilizing effects of PFN could not be attributed to differential binding of PFN. (c) 2005 Elsevier B.V. All rights reserved.
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