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.

Temperature-Dependent Expression of a CFP-YFP FRET Diacylglycerol Sensor Enables Multiple-Read Screening for Compounds That Affect C1 Domains

“…As expression levels increase, the FRET ratio of individual cells appears to converge to a consistent value. 1 The highest-expressing cells in both control (Fig. 1D, left panels) and room temperatureincubated ( Fig.…”

“…The generation of the DAGR cells, which are long-term transfectants of K562 cells that stably express the construct but were not cloned, was described previously, and both they and untransfected K562 cells were cultured as described. 1 We chose K562 cells because they are easily transfected to high levels of expression and grow quickly, facilitating the creation of long-term expressing lines. TALL-104 cells, which we used to analyze effects of putative C1 domain antagonists because we have experience analyzing PKC signaling in them, were obtained from the American Type Culture Collection (ATCC) and cultured as described previously.…”

“…All flow cytometry experiments were performed on a BD LSR Fortessa X-20 cell analyzer at the University of Connecticut's Flow Cytometry Facility as described previously. 1 The signals collected to characterize FRET signals were CFP (405 nm excitation, 450/50 nm emission, no dichroic mirror, 405 nm laser trigon), FRET (405 nm excitation, 525/50 nm emission, 505 LP dichroic mirror, 405 nm laser trigon), and YFP (488 nm excitation, 530/30 nm emission, 505 LP dichroic mirror, 488 nm laser octagon). Data were analyzed offline using FlowJo software (Tree Star, Inc., Ashland, OR).…”

“…We previously developed a multiplexed flow cytometry assay that used sensors for diacylglycerol (DAG), protein kinase C (PKC) activity, and extracellular signal-regulated kinase (ERK) activity, showing that the assay could identify compounds known to activate PKC. 1 There are a number of reasons why assays focused on this pathway are needed. Because they play important roles in the pathogenesis of Parkinson's disease, Alzheimer's disease, diabetes, cancer, and other important human diseases, [2][3][4][5][6] PKCs have long been targets of interest for drug development.…”

FRET Flow Cytometry-Based High Throughput Screening Assay To Identify Disrupters of Glucose Levels in Trypanosoma brucei

“…Although flow cytometers represent a powerful tool for high throughput screening, there are limited examples where they have been utilized for screening 19 . Similarly, FRET biosensors are rarely analyzed with flow cytometry even though flow cytometers are advantageous for analyzing a large number of cells expressing FRET sensors 20 …”