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 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.
D000841:Anisomycin / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015415:Biomarkers / Q000032:analysis*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002452:Cell Count / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016231:Fluorescence Polarization Immunoassay / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D008863:Microspheres; D025941:Protein Interaction Mapping / Q000379:methods*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015415:Biomarkers; D011518:Proto-Oncogene Proteins; D000841:Anisomycin; D004247:DNA; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases