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Multiplexed detection and quantification of apoptosis markers
Author(s) -
Brodey Mary Michael,
Zheng Wei,
Wang Jimin,
Fein Jeffrey A.,
Kevin Reagan
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.372
Subject(s) - apoptosis , jurkat cells , staurosporine , microbiology and biotechnology , cytochrome c , chemistry , poly adp ribose polymerase , immunoassay , immune system , biology , immunology , polymerase , biochemistry , signal transduction , t cell , antibody , dna , protein kinase c
The immune system is regulated and maintained through apoptosis, a normal process in which cell populations are deleted in response to self‐recognition, failure to bind MHC, and cytokine/growth factor withdrawal. To facilitate detection of apoptosis, we have developed a multiplexed sandwich immunoassay for the Luminex® platform that permits the simultaneous detection of several important biomarkers, including cytochrome c (a protein that normally resides within the intermitochondrial space that is released to the cytosol in response to apoptotic stimuli), cleaved caspase‐3 [175/176] (an important reporter for initiator caspase activation), and cleaved poly (ADP‐ribose) polymerase (PARP) [214/215] (an important reporter for caspase‐3 activation). To demonstrate the utility of this system, the three analytes were profiled in cell fractions prepared from Jurkat cells that were treated with 1 μM staurosporine to induce apoptosis and untreated controls. Significant changes in the profiles of the three analytes were detected within two hours of applying the stimulus. Linearity of dilution experiments performed in the validation of this assay system yielded R 2 values of 0.95 and higher.