A Flow Cytometric Immunophenotyping Approach to the Detection of Regulated Cell Death Processes

Abstract

The use of the Western Blot technique has been the gold standard to determine protein expression and to semi-quantitate this expression in cell lysates. The recent publication of a flow cytometric immunophenotyping method employing fluorescently labelled antibodies to the intracellular labelling of antigens involved in Regulated Cell Death (RCD) processes has allowed the detection of three of these processes simultaneously which gave clarity to the interpretation of the relationship between apoptosis, RIP1 dependent apoptosis and necroptosis. Flow cytometry can now immunophenotype necroptosis by virtue of the up-regulation of RIP3 with simultaneous estimations of the degree of classic apoptosis (Caspase-3+ve/RIP3ve) and of RIP1-dependent apoptosis (Caspase-3+ve/RIP3+ve) in live and dead cell populations. This approach for detecting multiple forms of cell death has been confirmed by the use of apoptosis and necroptosis blocking agents, zVAD and necrostatin-1 after treatment with etoposide or shikonin which induced apoptosis and necroptosis. The addition of anti-PARP and H2AX antibodies for the detection of parthanatos and DNA damage showed that double negative Caspase-3-ve/RIP3-ve cells detected in a previous study have undergone parthanatos or still display a negative phenotype for any cell death process. A Flow Cytometric Immunophenotyping Approach to the Detection of Regulated Cell Death Processes A Vossenkamper1, G Warnes2* 1Centre for Immunobiology, The Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary London University, 4 Newark Street, London E1 2AT, UK 2Flow Cytometry Core Facility, The Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary London University, 4 Newark Street, London E1 2AT, UK