HomeMeasurement of apoptosis by PARP
Apoptosis can be measured flow cytometrically by numerous assay's listed under Apoptosis. Cell cycle analysis can be used to measure late apoptosis by the detection of a SubG1 peak, see DNA fragmentation section. Cell cycle analysis can also be combined with the detection of cleaved PARP or Poly (ADP-ribose) polymerase-1. PARP is an 116 kDa enzyme involved DNA repair which during apoptosis is cleaved by active caspase-3. PARP can be detected flow cytometrically to show the presence of apoptosis after UV-irradiation or drug treatment.
Cell Cycle and detection of PARP
Flow cytometric detection of PARP can be achieved via tagging the protein with a fluorescently labelled antibody after treatment with BD CytoFix/Perm reagent. Cells can then be labelled with DNA binding dyes such as DAPI (1ug/ml) to show the stage of cell cycle the DNA breakages occur as shown by the presence of PARP signal. Colorectal carcinoma cell line C80 were treated under normoxic and hypoxic conditions for 3 days with and without drug treatment, see figure.
Parthantos a Regulated Form of Cell Death (RCD)
The parthantos is increased when cells undergo apoptosis or necroptosis after treatement with etoposide (induced apoptosis) or shikonin which induces both apoptosis and necroptosis depending upon the cell type. These levels can be modulated by pre-treatment with pan-caspase blocker and RIP protein blockers zVAD and necrostatin-1 respectively, see figure.
Publications
D Bergamashi, A Vossenkamper, WYJ Lee, P Wing, G Warnes. Simultaneous Polychromatic Flow Cytometric Detection of Multiple Forms of Regulated Cell Death. Apoptotis, 2019.
A Vossenkamper, G Warnes. A flow cytometric immunophenotyping approach to the detection of regulated cell death processes. Journal of Immunological Sciences, 2 (5) 2018.
TS MacFie, R Poulsom, A Parker, G Warnes, TBoitsova, A Nijhuis, N Suraweera, APoehlmann, JSzary, R Feakins, R Jeffery, RW Harper, AM Jubb, JO Lindsay, A Silver. DUOX2 and DUOXA2 form the predominant enzyme system capable of producing the reactive oxygen species H2O2 in active ulcerative colitis and are modulated by 5-aminosalicylic acid. Inflammatory Bowel Disease, Jan 2014.
