The Molecular Probes handbook. A guide to fluorescent probes and labeling technologies

Wiederschain, G. Ya.

doi:10.1134/s0006297911110101

Cited by 107 publications

(134 citation statements)

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“…As a commonly 70 used viability dye 11 , calcein enables determination of cell health as part of our cell tagging scheme without occupying an additional fluorescence channel. Commercially available calcein AM has seven protected carboxylic acids, all of which must be deprotected for maximal fluorescence 12 .…”

Section: Resultsmentioning

confidence: 99%

Spatially-Resolved Live Cell Tagging and Isolation Using Protected Photoactivatable Cell Dyes

Genshaft¹,

Ziegler²,

Tzouanas³

et al. 2020

Preprint

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Whether cultured in vitro or part of a complex tissue in vivo, a cell's phenotype and function are significantly influenced by dynamic interactions with its microenvironment. To explicitly examine how a cell's spatiotemporal activity impacts its behavior, we developed and validated a strategy termed SPACECAT-Spatially PhotoActivatable Color Encoded Cell Address Tags-to annotate, track, and isolate specific cells in a non-destructive, viability-preserving manner. In SPACECAT, a biological sample is immersed in a photocaged fluorescent molecule, and cells within a location of interest are labeled for further study by uncaging that molecule with user-patterned near-UV 10 light. SPACECAT offers high spatial precision and temporal stability across diverse cell and tissue types, and is compatible with common downstream assays, including flow cytometry and singlecell RNA-Seq. Illustratively, we leveraged this approach in patient-derived intestinal organoids, a spatially complex system less amenable to genetic manipulations, to select for crypt-like regions enriched in stem-like and actively mitotic cells. Moreover, we demonstrate its applicability and utility on ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model, uncovering spatially-biased gene expression patterns among immune cell subsets and identifying rare myeloid phenotypes enriched around tumor/healthy border regions. In sum, our method provides a minimally invasive and broadly applicable approach to link cellular spatiotemporal features and/or behavioral phenotypes with diverse downstream assays, enabling fundamental 20 insights into the connections between tissue microenvironments and biological (dys)function.

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Section: Resultsmentioning

confidence: 99%

Spatially-Resolved Live Cell Tagging and Isolation Using Protected Photoactivatable Cell Dyes

Genshaft¹,

Ziegler²,

Tzouanas³

et al. 2020

Preprint

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“…The increased fluorescence emission of dsDNA-bound PicoGreen depends on the structure and binding mode of PicoGreen [9,18]. PicoGreen has a 4-[[2,3dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-quinolini-um]+ core structure.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of PicoGreen variants for use in microscopy and flow cytometry

Khatoo

Yang

Gee

et al. 2019

Preprint

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PicoGreen is a fluorescent probe that binds dsDNA and forms a highly luminescent complex when compared to the free dye in solution. This unique probe is widely used in DNA quantitation assays but has limited application in flow cytometry and microscopy. Here we have investigated various PicoGreen variants for the ability to stain low amounts of cytosolic DNA present in many tumor cells. Analysis of stained cells by flow cytometry and fluorescent microscopy showed that certain variants improved the ability to stain low levels of cytosolic DNA when compared to the commercially available PicoGreen molecule.

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“…In nanotechnology, specifically with TiO 2 nanoparticles, Click chemistry was used for the preparation of hybrid nanomaterials [36–39]. In biology, Click chemistry reactions have been used for various cell biology and biomedical applications; the virtual absence of azide-carrying moieties in cells makes this an excellent approach for labeling with low background [40]. Specifically, several Click-based techniques were developed to investigate post-translational protein modifications to identify newly synthesized proteins and to examine DNA replication in proliferating cells [41–43].…”

Section: Discussionmentioning

confidence: 99%

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection

et al. 2017

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Titanium dioxide (TiO2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.

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The Molecular Probes handbook. A guide to fluorescent probes and labeling technologies

Cited by 107 publications

References 0 publications

Spatially-Resolved Live Cell Tagging and Isolation Using Protected Photoactivatable Cell Dyes

Spatially-Resolved Live Cell Tagging and Isolation Using Protected Photoactivatable Cell Dyes

Evaluation of PicoGreen variants for use in microscopy and flow cytometry

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection

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