Detection of functional cell surface perforin by flow cytometry.
How perforin (PFN) delivers the granzymes during cytotoxic granule mediated apoptosis remains a mystery. A major obstacle has been the inability to visualize PFN 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.
Metkar
Sunil S
SS
Division of Rheumatology, Department of Medicine, ENH Research Institute and Northwestern University Feinberg School of Medicine, Evanston, IL 60201, USA.
Wang
Baikun
B
Froelich
Christopher J
CJ
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D001681:Biological Assay; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D019812:Heparan Sulfate Proteoglycans / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019169:Jurkat Cells; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000276:immunology / Q000494:pharmacology; D054353:Perforin; D010539:Permeability / Q000187:drug effects; D052899:Pore Forming Cytotoxic Proteins
D000911:Antibodies, Monoclonal; D019812:Heparan Sulfate Proteoglycans; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin; D002118:Calcium