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Two optimized combination assays to examine apoptosis pathways in clinical samples.
Hollier M, Whistler T, Dawson C, Vernon SD
Two optimized combination assays to examine apoptosis pathways in clinical samples.
Cytometry Part A 2007;71A:675-685.
Summary
Apoptosis refers to the normal process by which cells in a multicellular organism die; it represents a form a programmed cell death. Apoptosis is an important biological phenomenon of normal life and defective apoptosis has been implicated in several diseases and has been hypothesized as having a role in the pathophysiology of CFS. Currently, there is no single assay that measures the main states of apoptosis, requiring that multiple assays be performed. This manuscript describes optimization of a series of combined assays that target specific stages of apoptosis and can be used in the typical clinical blood sample.
Abstract
A consequence of a number of diseases is an alteration in apoptosis. Currently, there is no single assay that measures the main stages of apoptosis, requiring that multiple assays be performed. This hinders studies on clinical samples that have limited cell numbers. Our objective was to combine and optimize assays that target specific stages of apoptosis for use in a typical clinical blood sample. Two flow cytometry assays were developed for use on peripheral blood mononuclear cells (PBMC) collected in two 8-ml tubes from a single draw. One measures caspase-12 activity, the level of active caspase-3 and DNA fragmentation. The second assesses depolarization of the mitochondria and phosphatidylserine externalization. Cell populations present within the samples were determined by flow cytometry. Apoptosis was validated by ELISA. Each assay was optimized for use with cell numbers and sample volumes typical of clinical blood samples. Each combination assay effectively distinguished apoptotic from non-apoptotic blood cells. This combined optimized method comprised of two independent assays makes it possible to assay the major pathways of apoptosis in addition to determining the blood cell subsets that are affected.
Page last modified on October 24, 2007
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