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Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model
Published on: April 16, 2014
Two optimized combination assays to examine apoptosis pathways in clinical samples
Mark Hollier1, Toni Whistler, Carolyn Dawson
1Chronic Viral Diseases Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30329, USA.
Background:
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.
Methods:
Two flow cytometric 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.
Results:
Each assay was optimized for use with cell numbers and sample volumes typical of clinical blood samples. Each combination assay effectively distinguished apoptotic from nonapoptotic blood cells.
Conclusions:
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.
Insights
This study developed a combined assay to measure key apoptosis stages in clinical blood samples. The optimized method efficiently analyzes apoptotic cells and affected blood cell subsets from limited samples.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Apoptosis (programmed cell death) is frequently altered in various diseases.
- Existing assays for apoptosis are not comprehensive, necessitating multiple tests.
- This limitation is particularly challenging for clinical studies with scarce cell samples.
Purpose of the Study:
- To develop and optimize a combined assay for assessing multiple apoptosis stages.
- To enable analysis of apoptosis in limited clinical blood samples.
Main Methods:
- Developed two flow cytometry-based assays for peripheral blood mononuclear cells (PBMC).
- Assays measure caspase-12 activity, active caspase-3, DNA fragmentation, mitochondrial depolarization, and phosphatidylserine externalization.
- Validated apoptosis detection using ELISA.
Main Results:
- Optimized assays for typical clinical blood sample volumes and cell counts.
- The combined assays successfully differentiated apoptotic from non-apoptotic blood cells.
- Identified affected blood cell subsets within the samples.
Conclusions:
- A novel, combined two-assay method allows simultaneous assessment of major apoptosis pathways.
- This approach is suitable for analyzing apoptosis in clinical blood samples, even with limited cell numbers.
- Facilitates the study of apoptosis in relation to specific blood cell populations.
