RESUMEN

Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections.

Asunto(s)

Técnicas Biosensibles/métodos , Citometría de Flujo/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/química , Humanos , Proteínas Luminiscentes/química , Toxinas de Lyngbya/química , Toxinas de Lyngbya/genética , Sistema de Señalización de MAP Quinasas/genética , Proteína Quinasa C/química , Proteína Quinasa C/genética , Transfección

RESUMEN

Extending observations on the immunogenicity of neo-antigens that arise in the course of oncogenesis and tumor progression, we suggest that somatic mutations affecting normal tissues also lead to generation of new epitopes. We hypothesize that, at least under inflammatory conditions, immune responses against such neo-antigens may lead to the elimination or functional impairment of normal cells, thus contributing to aging.