Analysis of apoptotic cells using Beadlyte suspension arrays

Here we describe a new approach to study apoptosis pathways using multiplex suspension arrays. Apoptosis was induced in Jurkat T cells using the protein synthesis inhibitor, anisomycin. Cells grown in 96-well plates were treated with anisomycin for up to 7 h, washed, and lysed in their respective we...

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Bibliographic Details
Published in:	BioTechniques Vol. 35; no. 3; pp. 624 - 629
Main Authors:	Rhyne, Paul W, Scull, Jason D, Stiles, Lynn M, Eisinger, Dominic P
Format:	Journal Article
Language:	English
Published:	England Taylor & Francis Group 01-09-2003
Subjects:	Anisomycin - pharmacology Apoptosis - drug effects Apoptosis - physiology Biomarkers - analysis Caspase 3 Caspases - metabolism Cell Count - methods Cell Culture Techniques - methods Cell Survival - drug effects Cell Survival - physiology DNA - metabolism Dose-Response Relationship, Drug Fluorescence Polarization Immunoassay - methods Humans Jurkat Cells - cytology Jurkat Cells - drug effects Jurkat Cells - physiology Microspheres Protein Interaction Mapping - methods Protein-Serine-Threonine Kinases Proto-Oncogene Proteins - metabolism Proto-Oncogene Proteins c-akt Reproducibility of Results Sensitivity and Specificity
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Summary:	Here we describe a new approach to study apoptosis pathways using multiplex suspension arrays. Apoptosis was induced in Jurkat T cells using the protein synthesis inhibitor, anisomycin. Cells grown in 96-well plates were treated with anisomycin for up to 7 h, washed, and lysed in their respective wells. Samples of each lysate were analyzed using Beadlyte suspension arrays consisting of total Akt/PKB, phosphorylated Akt/PKB, active caspase-3, and single-stranded DNA (ssDNA)-specific Beadmate microspheres and quantified with the X-MAP system. We found that phosphorylated Akt levels dropped dramatically with 2 h or more of anisomycin treatment, whereas active caspase-3 levels rose sharply with 2 h of treatment, signifying the onset of apoptosis. Longer incubation with anisomycin showed increases in ssDNA, which is consistent with the characteristic degradation of DNA that occurs in late-stage apoptosis. This approach demonstrates how apoptosis pathways can be studied from a small amount of sample without the use of more lengthy techniques such as immunoprecipitation or Western blotting. The suspension array is being expanded to measure many other intracellular proteins including posttranslational modifications and should prove to be extremely useful for studying apoptosis and other important cellular pathways.
ISSN:	0736-6205 1940-9818
DOI:	10.2144/03353pf01