Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening

Journal of Biomolecular Screening
Jaimee DoucetteAdam Zweifach

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 ...Continue Reading

References

Jun 6, 2000·Journal of Biomolecular Screening·J H ZhangK R Oldenburg
Dec 20, 2000·The Journal of Immunology : Official Journal of the American Association of Immunologists·L G PuenteH L Ostergaard
Jul 18, 2002·Nature Reviews. Drug Discovery·Margaret A Somerville
Jun 5, 2003·The Journal of Cell Biology·Jonathan D ViolinAlexandra C Newton
Nov 11, 2003·Trends in Pharmacological Sciences·ChengFeng Yang, Marcelo G Kazanietz
May 27, 2004·The American Journal of Pathology·Liusheng HePeter E Lipsky
Feb 25, 2006·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Sake van WageningenJoop H Jansen
Apr 15, 2006·Science·Ben N G GiepmansRoger Y Tsien
May 25, 2006·Chemistry & Biology·Amy E PalmerRoger Y Tsien
Jul 26, 2006·Proceedings of the National Academy of Sciences of the United States of America·James IngleseChristopher P Austin
Nov 28, 2006·Biochemical and Biophysical Research Communications·Daniel X JohanssonMats A A Persson
Dec 14, 2006·ACS Chemical Biology·Michael D AllenJin Zhang
Dec 26, 2006·The Journal of Biological Chemistry·Maya T KunkelAlexandra C Newton
Jan 3, 2008·Biochemical and Biophysical Research Communications·Takahiro Adachi, Takeshi Tsubata
Apr 17, 2008·Biochemical and Biophysical Research Communications·Arun T Pores-FernandoAdam Zweifach
May 2, 2008·Chemical Reviews·Marco Mank, Oliver Griesbeck
Nov 27, 2008·Proceedings of the National Academy of Sciences of the United States of America·Christopher D HarveyKarel Svoboda
Aug 18, 2009·Current Opinion in Chemical Biology·Nwe-Nwe Aye-HanJin Zhang
Nov 27, 2009·Journal of Biomolecular Screening·Anne KümmelChristian N Parker
Mar 10, 2010·Proceedings of the National Academy of Sciences of the United States of America·Matthew FosbrinkJin Zhang
May 13, 2010·Journal of Biomolecular Screening·Robert A Horton, Kurt W Vogel
Jun 29, 2010·Current Protocols in Molecular Biology·Charlene Depry, Jin Zhang
Apr 26, 2011·Current Topics in Medicinal Chemistry·Gustav Boije af GennäsElina Ekokoski
Oct 22, 2011·Nature Reviews. Molecular Cell Biology·Christopher M WelchKlaus M Hahn
Dec 23, 2011·Cancer Science·Kazuhiro AokiMichiyuki Matsuda
Nov 10, 2012·Pflügers Archiv : European journal of physiology·Charlene DepryJin Zhang
Mar 26, 2015·Journal of Biomolecular Screening·Bruce S Edwards, Larry A Sklar
Jul 3, 2015·International Journal of Molecular Sciences·Sean C WarrenPaul M W French

❮ Previous
Next ❯

Citations

Jul 22, 2018·SLAS Discovery·Mei Ding, Bruce S Edwards
Jun 16, 2021·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Jared HendersonRichard Eric Davis

❮ Previous
Next ❯

Methods Mentioned

BETA
FRET
transfection
flow cytometry
PMA
flow

Software Mentioned

Igor Pro
Plotly
Slidebook
DAGR
Excel
FlowJo
Open Lab
Improvision

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.

© 2022 Meta ULC. All rights reserved