title: Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.
abstract: 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.
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut at Storrs, Storrs, CT.
authors.forename: Jaimee
authors.initials: J
authors.lastname: Doucette
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut at Storrs, Storrs, CT.
authors.forename: Ziyan
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut at Storrs, Storrs, CT.
authors.forename: Rory J
authors.initials: RJ
authors.lastname: Geyer
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut at Storrs, Storrs, CT.
authors.forename: Melanie M
authors.initials: MM
authors.lastname: Barra
authors.affiliation: Division of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Connecticut at Storrs, Storrs, CT.
authors.forename: Marcy J
authors.initials: MJ
authors.lastname: Balunas
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut at Storrs, Storrs, CT adam.zweifach@uconn.edu.
authors.forename: Adam
authors.initials: A
authors.lastname: Zweifach
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D008235:Lyngbya Toxins / Q000737:chemistry / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000235:genetics; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D008235:Lyngbya Toxins; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; C019457:lyngbyatoxin A
keywords: cell-based assays; flow cytometry; fluorescence methods; kinases; multiplex assays and technology