Evelyn McGown, Ph.D., Jinfang Liao, M.D., Ph.D., Mary Kassinos, Irina Osetinsky and Jayne Hesley, Molecular Devices Corporation, 1311 Orleans Dr., Sunnyvale, CA 94089.
Fluorescent proteins have become enormously popular as tools for monitoring biological events in vivo. They can be cloned in a diverse range of cells and organisms, from bacteria and yeast, to plants and mammals. The fluorescent proteins are stable, have minimal toxicity and have the ability to generate visible fluorescence in vivo without the need for external cofactors. Thus they can be used as molecular tags or as independent reporters to visualize, track and quantify many different cellular processes, including protein synthesis and turnover, protein translocation, gene induction and cell lineage.
The various proteins have different colors, so they can be used in multiplexed assays. They can be monitored by fluorescent microscopy and flow cytometry. If there is no need to physically sort the cells or to monitor intracellular migration, microplate fluorometry offers a preferable, more convenient and higher-throughput detection system. Below, we show that the Analyst GT multimode reader from Molecular Devices can easily and noninvasively measure fluorescent proteins in living cells.
We obtained three HEK-293 cell lines from BD Biosciences Clontech, each stably transfected with a different fluorescent protein. The purposes of the study were: 1) to determine the optimal instrument settings for three cell lines, and 2) to prepare dilution series of each cell line to estimate lower limits of detection (LLD).
HEK-293 Cell Lines stably expressing fluorescent proteins were obtained from BD Biosciences Clontech:
AcGFPa variant of GFP cloned from Aequorea coerelescens (a jellyfish distinct from victoria)
ZsGreensimilar to GFP, but b