scholarly journals An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca 2+ and H + reveals new insights into ion signaling in plants

2021 ◽  
Author(s):  
Kunkun Li ◽  
Juan Prada ◽  
Daniel S. C. Damineli ◽  
Anja Liese ◽  
Tina Romeis ◽  
...  
Keyword(s):  
Dual Reporter ◽  
Ratio Imaging

Fluorescence ratio imaging of dynamic intracellular ionic signals

1988 ◽  
Vol 46 ◽  
pp. 42-43
Author(s):  
R. Y. Tsien ◽  
A. Minta ◽  
M. Poenie ◽  
J.P.Y. Kao ◽  
A. Harootunian
Keyword(s):  
Binding Sites ◽  
Living Cells ◽  
Molecular Engineering ◽  
Ph Indicators ◽  
Highly Sensitive ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Technical Advances ◽  
Indicator Dyes ◽  
Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

Ratio imaging of intracellular ion concentration

1992 ◽  
Vol 50 (2) ◽  
pp. 1160-1161
Author(s):  
Stephen R. Bolsover
Keyword(s):  
Individual Cell ◽  
Video Camera ◽  
Field Of View ◽  
Ion Concentration ◽  
Fluorescence Signal ◽  
Ion Imaging ◽  
Ratiometric Fluorescence ◽  
Ratio Imaging ◽  
The Mean ◽  
The Many

The field of intracellular ion concentration measurement expanded greatly in the 1980's due primarily to the development by Roger Tsien of ratiometric fluorescence dyes. These dyes have many applications, and in particular they make possible to image ion concentrations: to produce maps of the ion concentration within living cells. Ion imagers comprise a fluorescence microscope, an imaging light detector such as a video camera, and a computer system to process the fluorescence signal and display the map of ion concentration.Ion imaging can be used for two distinct purposes. In the first, the imager looks at a field of cells, measuring the mean ion concentration in each cell of the many in the field of view. One can then, for instance, challenge the cells with an agonist and examine the response of each individual cell. Ion imagers are not necessary for this sort of experiment: one can instead use a system that measures the mean ion concentration in a just one cell at any one time. However, they are very much more convenient.

scholarly journals Establishment of MHHi001-A-5, a GCaMP6f and RedStar dual reporter human iPSC line for in vitro and in vivo characterization and in situ tracing of iPSC derivatives

2021 ◽  
Vol 52 ◽  
pp. 102206
Author(s):  
Alexandra Haase ◽  
Tim Kohrn ◽  
Veronika Fricke ◽  
Maria Elena Ricci Signorini ◽  
Merlin Witte ◽  
...  
Keyword(s):  
Ipsc Line ◽  
Dual Reporter ◽  
In Vivo Characterization ◽  
Human Ipsc

Rescue of dual reporter-tagged parainfluenza virus 5 as tool for rapid screening of antivirals in vitro

2021 ◽  
pp. 109154
Author(s):  
Fuxiao Liu ◽  
Qianqian Wang ◽  
Hu Shan
Keyword(s):  
Parainfluenza Virus ◽  
Rapid Screening ◽  
Dual Reporter ◽  
Parainfluenza Virus 5

scholarly journals GATA-targeted compounds modulate cardiac subtype cell differentiation in dual reporter stem cell line

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mika J. Välimäki ◽  
Robert S. Leigh ◽  
Sini M. Kinnunen ◽  
Alexander R. March ◽  
Ana Hernández de Sande ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Reporter Gene ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Cell Fate Decisions ◽  
Dual Reporter ◽  
Gene Regulatory ◽  
Reporter Gene Assays

AbstractBackgroundPharmacological modulation of cell fate decisions and developmental gene regulatory networks holds promise for the treatment of heart failure. Compounds that target tissue-specific transcription factors could overcome non-specific effects of small molecules and lead to the regeneration of heart muscle following myocardial infarction. Due to cellular heterogeneity in the heart, the activation of gene programs representing specific atrial and ventricular cardiomyocyte subtypes would be highly desirable. Chemical compounds that modulate atrial and ventricular cell fate could be used to improve subtype-specific differentiation of endogenous or exogenously delivered progenitor cells in order to promote cardiac regeneration.MethodsTranscription factor GATA4-targeted compounds that have previously shown in vivo efficacy in cardiac injury models were tested for stage-specific activation of atrial and ventricular reporter genes in differentiating pluripotent stem cells using a dual reporter assay. Chemically induced gene expression changes were characterized by qRT-PCR, global run-on sequencing (GRO-seq) and immunoblotting, and the network of cooperative proteins of GATA4 and NKX2-5 were further explored by the examination of the GATA4 and NKX2-5 interactome by BioID. Reporter gene assays were conducted to examine combinatorial effects of GATA-targeted compounds and bromodomain and extraterminal domain (BET) inhibition on chamber-specific gene expression.ResultsGATA4-targeted compounds 3i-1000 and 3i-1103 were identified as differential modulators of atrial and ventricular gene expression. More detailed structure-function analysis revealed a distinct subclass of GATA4/NKX2-5 inhibitory compounds with an acetyl lysine-like domain that contributed to ventricular cells (%Myl2-eGFP+). Additionally, BioID analysis indicated broad interaction between GATA4 and BET family of proteins, such as BRD4. This indicated the involvement of epigenetic modulators in the regulation of GATA-dependent transcription. In this line, reporter gene assays with combinatorial treatment of 3i-1000 and the BET bromodomain inhibitor (+)-JQ1 demonstrated the cooperative role of GATA4 and BRD4 in the modulation of chamber-specific cardiac gene expression.ConclusionsCollectively, these results indicate the potential for therapeutic alteration of cell fate decisions and pathological gene regulatory networks by GATA4-targeted compounds modulating chamber-specific transcriptional programs in multipotent cardiac progenitor cells and cardiomyocytes. The compound scaffolds described within this study could be used to develop regenerative strategies for myocardial regeneration.

scholarly journals Tumorigenicity of hypoxic respiring cancer cells revealed by a hypoxia-cell cycle dual reporter

2014 ◽  
Vol 111 (34) ◽  
pp. 12486-12491 ◽  
Author(s):  
A. Le ◽  
Z. E. Stine ◽  
C. Nguyen ◽  
J. Afzal ◽  
P. Sun ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cancer Cells ◽  
Dual Reporter

scholarly journals Dual reporter genes enabling cell tracing with viable and reliable selection of various cell types

2006 ◽  
Vol 28 (22) ◽  
pp. 1865-1865
Author(s):  
C. N. Hwang ◽  
S. Hong ◽  
S. S. Choi ◽  
K. S. Lee ◽  
S. S. Park ◽  
...  
Keyword(s):  
Reporter Genes ◽  
Cell Types ◽  
Dual Reporter ◽  
Cell Tracing ◽  
Selection Of

Synthesis and characterization of dual-wavelength Cl−-sensitive fluorescent indicators for ratio imaging

1999 ◽  
Vol 276 (3) ◽  
pp. C747-C757 ◽  
Author(s):  
Sujatha Jayaraman ◽  
Joachim Biwersi ◽  
A. S. Verkman
Keyword(s):  
First Generation ◽  
Spectral Characteristics ◽  
Dual Wavelength ◽  
Reduced Form ◽  
Excitation Wavelength ◽  
Transmembrane Conductance Regulator ◽  
Fluorescent Indicators ◽  
3T3 Fibroblasts ◽  
Ratio Imaging ◽  
Ratiometric Measurement

The fluorescence of quinolinium-based Cl− indicators such as 6-methoxy- N-(3-sulfopropyl)quinolinium (SPQ) is quenched by Cl− by a collisional mechanism without change in spectral shape. A series of “chimeric” dual-wavelength Cl− indicators were synthesized by conjugating Cl−-sensitive and -insensitive chromophores with spacers. The SPQ chromophore (N-substituted 6-methoxyquinolinium; MQ) was selected as the Cl−-sensitive moiety [excitation wavelength (λex) 350 nm, emission wavelength (λem) 450 nm]. N-substituted 6-aminoquinolinium (AQ) was chosen as the Cl−-insensitive moiety because of its different spectral characteristics (λex 380 nm, λem 546 nm), insensitivity to Cl−, positive charge (to minimize quenching by chromophore stacking/electron transfer), and reducibility (for noninvasive cell loading). The dual-wavelength indicators were stable and nontoxic in cells and were distributed uniformly in cytoplasm, with occasional staining of the nucleus. The brightest and most Cl−-sensitive indicators were α-MQ-α′-dimethyl-AQ-xylene dichloride and trans-1,2-bis(4-[1-α′-MQ-1′-α′-dimethyl-AQ-xylyl]-pyridinium)ethylene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm (Cl− sensitive; Stern-Volmer constants 82 and 98 M−1) and 565 nm (Cl−insensitive). Cystic fibrosis transmembrane conductance regulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQ by hypotonic shock or were labeled with its uncharged reduced form (octahydro-bis-DMXPQ) by brief incubation (20 μM, 10 min). Changes in Cl− concentration in response to Cl−/nitrate exchange were recorded by emission ratio imaging (450/565 nm) at 365-nm excitation wavelength. These results establish a first-generation set of chimeric bisquinolinium Cl− indicators for ratiometric measurement of Cl− concentration.

Development of a dual reporter screening assay for distinguishing the inhibition of HIV Tat-mediated transcription from off-target effects

2017 ◽  
Vol 249 ◽  
pp. 1-9 ◽  
Author(s):  
YoungHyun Shin ◽  
Byeong-Sun Choi ◽  
Kyung-Chang Kim ◽  
Chun Kang ◽  
Kisoon Kim ◽  
...  
Keyword(s):  
Screening Assay ◽  
Hiv Tat ◽  
Dual Reporter ◽  
Target Effects

Analysis and characterization of high-resolution and high-aspect-ratio imaging fiber bundles

2015 ◽  
Vol 54 (32) ◽  
pp. 9422 ◽  
Author(s):  
Nojan Motamedi ◽  
Salman Karbasi ◽  
Joseph E. Ford ◽  
Vitaliy Lomakin
Keyword(s):  
High Resolution ◽  
Aspect Ratio ◽  
High Aspect Ratio ◽  
Fiber Bundles ◽  
Ratio Imaging
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